What controls specific gene silencing during cell differentiation?

What controls specific gene silencing during cell differentiation?

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I am intrigued by the fact that all cells of our body use the same DNA. How do the cells differentiate during the post fertilisation divisions?

I read about gene silencing, which can be an answer to this. But still I do not understand how the cells decide which gene it is supposed to silence, by making the micro RNA.

Consider a zygote that is beginning to divide. I would assume that the chemical and physical context of each daughter cells would be more or less similar in beginning. Then how can there be a differentiation? Is there some supervising authority present in the zygote that decides what to make out of each daughter cell?

The cells differentiation during post fertilization period is govern by a set of regulatory genes called Homeotic genes.These are genes that "select" the identity of entire segments or structures in the bodies of developing organisms. These gene encodes a transcription factor that is expressed in a specific region of the organism starting in its early development i.e. embryo stage. The transcription factors change the expression of target genes to enact the genetic “program” that's right for each segment.

The homeotic transcription factors shown in the diagram above[homology between Hox genes in mice and humans] contain a DNA-binding protein region called the homeodomain, which is encoded by a segment of DNA, called the homeobox.The animal genes containing homeobox sequences are specifically referred to as Hox genes. This family of genes is responsible for determining the general body plan, such as the number of body segments of an animal, the number and placement of appendages, and animal head-tail directionality.

In addition to this genes, the early developmental cascade include the following genes:

  • Maternal effects genes:These are genes whose mRNAs are placed in the egg cell by the mother before fertilization. Some of the mRNAs are “tied” to the head or tail end of the embryo and are responsible for setting up the head-tail polarity. The maternal effects genes encode regulators of transcription or translation that control each other as well as other genes.
  • Gap genes: They are activated through interactions between the protein products of the maternal effects genes, and they also regulate each other. They're responsible for defining large, multi-segment regions in many organisms.
  • Pair-rule genes: These are turned on by interactions between gap genes, and their expression patterns are refined by interactions with one another. They appear in multiple “stripes” along the embryo, similar in pattern to the segments of the adult organism.When a pair-rule gene is missing due to mutation, there is a loss of structures in the segment regions where the gene is normally expressed.

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Activation and deactivation of different genes commands the process of cell differentiation. Gene expression responsible for cell differentiation is controlled by intrinsic and extrinsic signals. This regulated signalling from inside and outside the cell is responsible for embryonic development.

Environment around the cell, such as small molecules, proteins, temperature and oxygen control the gene expression. Cellular communication takes places to decide the fate of particular cell by interplay of signals between the proteins synthesized around the cell. These proteins can be morphogens, growth factors or cytokines. This extrinsic signalling sets off intercellular signalling that stimulates expression of genes. Alteration in gene expression by turning gene on or off, regulates the production of gene product.

Gene expression is regulated intrinsically by modifying DNA. DNA and chromatin are altered chemically. Change in chromatin effects gene expression by controlling the binding of genes to transcription factors. These epigenetic chemical modifications are known as DNA methylation and histone modification. Chromatin modification plays important role in gene expression during cell development. For example, proteins responsible for chromatin modification play important role in muscle cell differentiation. Transcription factors MyoD and MEF regulate enzymes responsible for chromatin modification, such as histone acetyltransferases and deacetylases.


Chromatin modification helps in continuous gene expression which is required during cell differentiation. The gene silencing involved in embryogenesis, promotes cell development into mature cell types. This silencing of gene is done by making gene inaccessible to transcription machinery, and when genes are needed again in adult cell type the chromatin modification opens DNA and make it available for transcription.

Embryonic cell types have particular regions for chromatin modification that regulate gene expression required for embryonic development. These regions can modify gene expression by activating or silencing genes.

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Hematopoiesis is controlled by interactions between hematopoietic stem cells and their microenvironment. These interactions influence retention of stem cells in specific niches, and stem and progenitor cell expansion, lineage divergence and differentiation [1]. Adhesion molecules are major regulators of cell-cell interactions and they influence multiple aspects of hematopoiesis [1–4]. Indeed, antibodies against various adhesion molecules including VLA-4 and VCAM-1 inhibit the ability of hematopoietic stem cells to populate the bone marrow of irradiated mice [5], and gene knock-out studies of integrins have shown their critical role in homing and colonization of late-stage primary hematopoietic organs such as the embryonic liver [6, 7]. More recently, N-cadherin expression has been implicated in retention of hematopoietic stem cells in the bone marrow niche [8–10] although this claim is not supported by other studies [11]. In contrast to their role in homing, our understanding of adhesion molecule biology in lineage commitment and differentiation is poorly defined.

Hematopoietic cell antigen, also known as activated leukocyte cell adhesion molecule (ALCAM/CD166), is a member of the immunoglobulin super-family. It is expressed on the surface of the most primitive hematopoietic cells in human fetal liver and fetal and adult bone marrow [12]. Other studies have found ALCAM expression on subsets of stromal cells in the para-aortic mesoderm and other primary sites of hematopoiesis in the human embryo [13]. ALCAM-mediated interactions are important during neural development [14], maturation of hematopoietic stem cells in blood forming tissues [12, 15], immune responses [16] and in tumor progression [17]. Anti-ALCAM antibodies inhibit myeloid colony formation in vitro by mechanism that remains unknown [18]. We showed previously that ALCAM is involved in transmigration of monocytes across endothelial monolayers [19]. More recent in vivo studies have shown that ALCAM is essential for monocyte migration across the blood-brain barrier [20]. Other studies indicate the interaction of ALCAM on dendritic cells with the T-cell ligand CD6 is required for optimal T-cell activation [21]. While these studies highlight ALCAM's role in mature and activated leukocyte cell biology, there is currently no information on ALCAM's role in hematopoietic progenitor cell biology.

In this study, we examined ALCAM expression in human hematopoietic cell lines. The ALCAM gene was cloned and functionally characterized in K562 cell lines. The influence of ALCAM on megakaryocytic differentiation of K562 cells was investigated.


Transcriptional pausing is the phenomenon in which genes experience initiation of transcription without elongation. Initially thought to occur only in rare instances such as Drosophila heat shock genes (reviewed in [1]), Krumm et al proposed that pausing might be a more general mechanism based on detailed analysis of the mouse c-myc gene [2]. More recent studied using chromatin immunoprecipitation followed by chip hybridization (ChIP-chip) or deep sequencing (ChIP-seq) has revealed that transcriptional pausing does, indeed, occur at a large fraction of genes in mammalian [1], [3], [4], [5], [6], [7] and Drosophila [8], [9] cells. Non-productive short transcripts in both the sense and antisense [10], [11] directions have been described at many genes, and many developmental regulatory genes appear to be paused in both pluripotent and differentiated cell types. In contrast, constitutively active housekeeping genes generally do not exhibit transcriptional pausing [12]. This suggests that transcriptional pausing is a widespread mechanism of controlling cell-type specific gene expression programs.

Despite these advances, the physiological significance of transcriptional pausing is still unclear. We noted that most studies of pausing, to date, have focused on cells in the steady state. We hypothesized that pausing may be important for permitting cells to rapidly change gene expression levels in response to environmental cues. The differentiation of pluripotent stem cells towards more committed cells provides a human model system to study one of the most dramatic changes of cellular states. We sought to understand how the transcription apparatus is dynamically regulated as new patterns of gene expression are established during differentiation. To test the hypothesis that transcriptional pausing is a key transition state for gene expression, we used a directed system to differentiate human embryonic stem cells to early mesoderm [13] and quantified the flow of protein-coding loci between active, paused and silent states using a combination of chromatin immunoprecipitation and 3′ transcript analysis.


In this study, we determined the dynamics of gene silencing on the (future) Xi by allele-specific RNA-seq during differentiation of female ESCs. We optimized the allele-specific RNA-seq mapping by GSNAP [46] in an efficient and straightforward procedure, thereby obtaining unbiased high-resolution gene expression profiles from both alleles. The silencing kinetics for individual genes during XCI reveals a linear component in the propagation of inactivation over the Xi. This is supported by the increase in distance of four kinetic clusters associated with gene silencing, as well as by the high ratio of gene silencing for genes near the XIC at very early stages of XCI. The escape from XCI of three regions very distal from the XIC, in differentiated ES_Tsix-stop ESCs as well as in NPCs, might be a consequence of incomplete linear spread. It has been shown that XCI-mediated silencing can only occur in a short time-window of embryonic development/differentiation also referred to as the “window of opportunity” [53]. As a consequence, cells that do not complete XCI within this time frame might fail to inactivate parts of the X chromosome that are at greater distance from the XIC and hence silenced late. The NPCs used in the current study, as well as the NPCs generated by Gendrel et al. [52] in which the escape regions are also present, have been derived from ES_Tsix-stop ESCs [35]. During the extensive in vitro differentiation towards NPCs, a subset of ESCs might have completed XCI (NPC_129-Xi), while in other cells the XCI process remains incomplete (*NPC_129-Xi and NPC_Cast-Xi). In the latter cells, parts of the Xi remain active, as they are not silenced during the window of opportunity. Apparently, the activity of the non-silenced genes on the Xi is tolerated in the NPCs, although it might affect cell viability as we noticed that the *NPC_129-Xi and NPC_Cast-Xi NPC lines show increased doubling times compared with NPC_129-Xi.

If indeed the escape regions result from incomplete XCI during the window of opportunity, their localization at regions very distal to the XCI would further support a linear model of propagation of XCI from the XIC over the (future) Xi. However, similar to what has been shown for imprinted XCI of the paternal Xi during early mouse development [54], linearity clearly only explains part of the silencing dynamics we observe. Various genes near the XIC are inactivated late and show no signs of silencing at early time points, while other genes very distal from the XIC are silenced early. Therefore, other components such as spatial organization of the X chromosome, TADs (as discussed below) and local chromatin environment likely play important roles in the silencing dynamics on the Xi. Indeed, it has been shown that the earliest regions containing enriched occupancy of Xist are spread across the entire linear X chromosome, but do have spatial proximity to the XIC [17, 18]. Furthermore, also the level of gene expression affects the kinetics of XCI silencing, as we observe that highly expressed genes show a slight but significant delay in silencing compared with lowly expressed genes. This might be caused by the fact that it takes longer for these highly expressed genes to alter the local chromatin environment by depositing marks associated with silencing, such as H3K27me3 [22, 55, 56]. On the other hand, the stability of the various RNAs also influences the kinetics of X-linked silencing during XCI. Stable RNAs have a longer half-life and will, therefore, show slower silencing dynamics in our analysis. A recent study investigating stability of X-linked transcripts showed an overall increase in half-life of X-linked transcripts versus autosomal transcripts [57, 58]. Amongst X-linked transcripts, the half-life varied between 2 and 15 h, with the median half-life being 6 h. Since this time frame is much shorter than the 8-day course of EB differentiation, stability of RNA likely has little influence on the clustering we performed (Fig. 4). Rather, the clustering has been dictated by silencing of transcription on the chromatin.

The three escape regions identified in the current study (Figs. 5 and 6) largely correspond to TADs as characterized in the undifferentiated female ESCs. Together with the observation that the escape clusters in human closely correlate with TADs, this suggests a functional role for the TADs during XCI. Previously, TADs have been implicated in the regulation of XCI within the XIC, with the promoters of Tsix and Xist being present in neighboring TADs with opposite transcriptional fates [59]. Furthermore, it has been shown that TADs align with coordinately regulated gene clusters [59]. The current observation that the regions escaping XCI correspond to TADs suggests that genes within TADs are co-regulated to induce silencing in a domain-type fashion during XCI. This would imply that TADs are the functional compartments in the higher order chromatin structure that are targeted for inactivation during initiation of XCI. Once targeted, silencing might be propagated within the TAD such that the associated genes become inactivated. How this would work remains to be resolved, but the functional mechanisms might resemble those acting in long range epigenetic silencing (LRES) by which large regions (up to megabases) of chromosomes can be co-coordinately suppressed [60].

Together, the dynamics of XCI we observe fit with previously proposed biphasic models in which secondary spread of inactivation occurs via so-called relay elements, way stations or docking stations, the nature of which still remains elusive [18, 21, 22, 61] (see Ng et al. [62] for a recent review). Our study suggests that TADs are the primary targets during propagation of XCI, after which secondary spread occurs within TADs. Such involvement of TADs in XCI is likely to be very early during the inactivation process, as it has been shown that the Xi has a more random chromosomal organization at later stages in which global organization in TADs is reduced and specific long-range contacts within TADs are lost [35, 59, 63]. An interesting possibility to further investigate the role of TADs during inactivation of the (future) Xi is to investigate gene silencing within TADs during XCI — for example, during the EB formation time course we performed. However, the current resolution of allele-specific RNA-seq lacks resolution for such analysis, mainly due to (i) the limited number of polymorphic sites available to distinguish both alleles and (ii) the very high depth of sequencing necessary to obtain reliable allele specific calls for lowly expressed genes (which by definition will have low coverage over polymorphic sites). For the current study we obtained allelic information for 259 X-linked genes over the EB differentiation time course, while the X chromosomes consists of 124 TADs (Additional file 7: Table S5). This average number of genes per TAD is insufficient to study expression dynamics within TADs.

Besides the genes within the escape regions, none of the remaining genes on the X chromosome are present in clusters of contiguous escape genes. Also, other escape genes co-occupy the TAD in which they are localized with genes that are subject to XCI. Therefore, the escape of genes outside the escape regions is likely instructed by epigenetic features other than TADs. This might also be the case for the well-known escape gene Ddx3x, which is part of escape region 2 but not part of the TAD that is associated with this region. Next to the escape genes reported in Table 1, we detect some (very) low level escape in all three NPC lines: an additional

50 genes show <10 % contribution of the Xi to the total expression of a gene (in most cases <1 %) mostly corresponding to five or less sequence tags (Additional file 5: Table S4). A recent study reporting a similar finding in NPCs proposed that this is associated with a relaxation in the epigenetic state in NPCs as well as in neural stem cells in brain tissue [52], suggesting that reactivation from the Xi can occur for these genes. Also for individual escape genes such as Kdm5c, it has been reported that they were initially silenced at the onset of XCI, after which they are reactivated later during development from the Xi [38, 50]. However, the majority of escape genes in the NPCs identified in the current study already (largely) escape silencing during establishment of XCI, as they are present in the “late” or “not silenced” kinetic clusters 3 or 4 in the female EB differentiations. This suggests that escape genes are already excluded from XCI from the start, and that most of these escape genes, therefore, likely contain (epi)genetic features that exclude them from being silenced during propagation of XCI.

By determining global levels of gene expression at different stages of differentiation and development, our data furthermore provide insight into the dynamics of dosage compensation between the X chromosome and autosomes. In ESCs, the mean level of X-linked gene expression in female and male is 1.50- and 0.86-fold higher, respectively, than expression from autosomal genes (Additional file 1: Figure S1 Fig. 2c). Compared with ESCs, expression of female X-linked genes in epiblast stem cells (EpiSCs) is reduced, while expression of male X-linked genes is increased. Autosomal expression is relatively stable between female and male ESCs and EpiSCs. This results in very similar levels of expression between autosomal and X-linked genes in male and female EpiSCs (Additional file 1: Figure S1), in line with previous observations by Lin et al. [23]. Very similar dynamics are obtained during EB differentiation, during which X-linked genes are slightly upregulated from the Xa in female (Fig. 2c) and the single X chromosome in male ESCs (Fig. 3b, right panel). This suggests that full dosage compensation in differentiated cell types is achieved by upregulation of the genes on the Xa in female and the single X chromosome in male cells during early embryonic development.

Key Points

During cellular differentiation, only specific subsets of genes are required to carry out a cell's specialized function. The remainder are silenced. Inactive regions of the chromosome are packaged into transcriptionally inactive heterochromatin, mediated by histone deactylation these regions are unique to each differentiated cell type.

Gene potentiation is a prerequisite to gene activation. This opens the chromatin structure so that DNA is accessible to the activator proteins required for transcription. Histone acetylation is required to mediate this chromatin opening.

Chromosomes and genes are organized into specific nuclear zones, termed chromosome territories within this, potentially active regions are located at the periphery. These territories occupy non-random positions in the interphase nucleus, specific to each cell type. Changes in nuclear architecture are proposed to occur during differentiation.

Nuclear functions such as transcription also occur in specific compartments of the nucleus. Therefore, nuclear architecture may allow active genes to localize to regions that are permissive for transcription.

Evidence from Drosophila melanogaster and mammals indicates that positioning of a gene near centromeric heterochromatin often promotes gene silencing and likewise, that sequestration of a gene into a permissive compartment often allows the stably inherited chromatin opening of a locus.

Modification of chromatin structure over large regions is proposed to be initiated by assembly of proteins on cis-acting elements called silencer elements. Proteins that bind to these elements include Sir proteins in yeast, and the Polycomb proteins in Drosophila. This repressive structure is proposed to propagate along the chromosome.

Studies indicate that enhancer elements can counteract these silencing events. Binding of enhancer elements is proposed to recruit the gene to a region in the nucleus that is rich in the transcription factors and histone acetylases required to activate transcription.

Cancer is associated with disruption of gene expression patterns and global disorganization of chromosome organization within the nucleus.


Primordial germ cells (PGCs) are the founding population of cells that will ultimately give rise to the mature gametes. Unlike organisms that have a mosaically determined germline, PGCs in the mouse embryo are specified by an inductive mechanism that requires the presence of several bone morphogenetic proteins (BMPs) emanating from the surrounding somatic cells(Fujiwara et al., 2001 Lawson et al., 1999 Ying et al., 2000 Ying and Zhao, 2001). PGCs can first be detected in the extra-embryonic mesoderm at 7.25 days post-coitus(dpc) (Ginsburg et al., 1990). By 8.5 dpc, PGCs enter the embryo proper and actively migrate through the hindgut endoderm, colonizing the developing gonads between 10.5 and 11.5 dpc. During this time, PGCs proliferate from an initial population of 45 cells at 7.5 dpc to 25,000 cells at 13.5 dpc when proliferation ceases(Tam and Snow, 1981). Sexual differentiation of the germline begins at 13.5 dpc with female germ cells entering prophase I of meiosis. Within the male gonad, a signal thought to originate from the testis cords prevents entry into meiosis and male PGCs enter a mitotic arrest by 14.5 dpc(McLaren, 1983). Prior to these changes, male and female PGCs are sexually indifferent, capable of following either the male or female pathway(McLaren and Southee,1997).

Shortly after PGCs enter the urogenital ridges, both male and female germ cells undergo a common set of changes independent of sexual differentiation. Changes in cell morphology and cell-adhesion properties occur as the germ cells transition to a nonmigratory state(De Felici et al., 1992 Donovan et al., 1986 Garcia-Castro et al., 1997). Male and female PGCs also cease proliferating, have decreased potential to form pluripotent stem cell lines (Matsui et al., 1992 McLaren,1984 Resnick et al.,1992), and undergo a wave of apoptosis(Coucouvanis et al., 1993 Wang et al., 1998). These differentiation events are accompanied by changes in gene expression as some germ cell marker genes, such as Tnap (Akp2 - Mouse Genome Informatics) and Zfp148, are downregulated(Donovan et al., 1986 Hahnel et al., 1990 Takeuchi et al., 2003). Other genes, including Mvh (Ddx4 - Mouse Genome Informatics), Scp3 (Sycp3 - Mouse Genome Informatics), Dazl,Mageb4 and Gcna1 are upregulated during this time(Cooke et al., 1996 Di Carlo et al., 2000 Fujiwara et al., 1994 Osterlund et al., 2000).

In addition to the differentiation events mentioned above, PGCs mediate two essential epigenetic processes. First, female PGCs reactivate their silenced X chromosome, thereby ensuring that each oocyte carries an active X chromosome(Monk and McLaren, 1981 Tam et al., 1994). Interestingly, the ability to reactivate the inactive X chromosome is not confined to female germ cells, as XXY male germ cells also possess this reactivation capability (Mroz et al.,1999). Second, migratory germ cells carry parent-of-origin-specific imprinting marks and high levels of allele-specific methylation that contribute to monoallelic expression in migratory PGCs. These differentially methylated regions become hypomethylated as PGCs colonize the gonads, leading to a loss of imprinting and biallelic gene expression(Hajkova et al., 2002 Lee et al., 2002 Szabo et al., 2002). However,this wave of demethylation is not restricted to imprinted loci and genes of the X chromosome, as several non-imprinted genes and repetitive sequences also show decreased methylation at this time(Hajkova et al., 2002 Lane et al., 2003 Lees-Murdock et al.,2003).

We have been investigating regulatory mechanisms underlying postmigratory germ cell differentiation. Several studies suggest that continuing PGC development is regulated by a cell intrinsic program rather than by inductive signals from the gonads. PGCs located in ectopic locations enter meiosis and initiate expression of the postmigratory marker GCNA1 on schedule without exposure to the urogenital ridges (Wang et al., 1997). Embryonic stem cells have been shown to differentiate to form PGC-like cells that can go on to form cells resembling both oocytes and spermatocytes, further demonstrating that PGC differentiation can occur independently of the gonadal environment(Geijsen et al., 2004 Hubner et al., 2003 Toyooka et al., 2003). Last,cessation of germ cell proliferation has also been suggested to be cell intrinsic (Ohkubo et al.,1996).

We previously tested the potential of premigratory germ cells to differentiate in culture and reported that 8.5 dpc premigratory PGCs in feeder culture can differentiate to express GCNA1 on the correct temporal schedule(Richards et al., 1999). Surprisingly, the rate of differentiation in culture increased when PGCs were exposed to the DNA demethylating agent 5-azacytidine or the histone deacetylase inhibitor trichostatin A(Maatouk and Resnick, 2003). This suggests that epigenetic mechanisms may contribute to the regulation of germ cell differentiation.

Here, we further investigate the role of DNA methylation in the process of PGC differentiation. We present evidence that several postmigratory germ cell-specific genes are demethylated in germ cells as they colonize the genital ridges and that DNA demethylation controls the temporal expression of these genes in vivo. In addition, we show that these postmigratory germ cell-specific genes are ectopically expressed in DNA methyltransferase mutant embryos, suggesting that DNA methylation is a mechanism of silencing germ cell-specific genes in somatic tissues. These results provide the first in vivo evidence of tissue-specific embryonic gene regulation mediated by dynamic changes in DNA methylation.

Gene regulation by H3K9me3 in development

The role of H3K9me3 in gene regulation in somatic tissues

In metazoans, gametogenesis and early embryogenesis are accompanied by extensive epigenetic reprogramming during which most chromatin marks, including H3K9me3, are erased to grant totipotency to the zygote and are later re-established. H3K9me3 re-establishment in somatic tissues is essential for normal developmental progression in Drosophila and mammals. Interestingly, loss of silencing effectors that primarily control H3K9me3 deposition at constitutive heterochromatin leads to less severe phenotypes than disruption of H3K9me3 deposition outside constitutive heterochromatin. For example, mutant mice double null for the two SUV39 paralogs, which primarily localize to centromeric regions, display chromosomal instability and multiple defects, yet some animals survive to adulthood (Peters et al., 2001). Conversely, upon loss of SetDB1/ESET, which is primarily involved in H3K9me3 deposition outside constitutive heterochromatin, the inner cell mass of the pre-implantation embryo fails to form properly, leading to pre-implantation lethality (Dodge et al., 2004). Likewise, in Drosophila, Su(var)3-9 null mutants are viable and fertile (Tschiersch et al., 1994), while SetDB1 loss-of-function mutations are homozygous lethal (Seum et al., 2007). Loss of several members of the HP1 family of H3K9me3 readers, including the Drosophila Su(var)2-5/HP1a and the mouse Cbx1/HP1β, also result in developmentally lethal phenotypes (Aucott et al., 2008 Eissenberg et al., 1990 Eissenberg et al., 1992 Kellum and Alberts, 1995), highlighting the essential role of H3K9me3 in early development.

H3K9me3 in the early embryo and ESCs

Most current knowledge about the role of H3K9me3 in early somatic development comes from studies in murine systems, which have shown that gene silencing by H3K9me3 is particularly important during pre-implantation embryonic development, ESC self-renewal, cell differentiation and cell lineage commitment.

It is well established that there is a complex interplay between H3K9me and DNA methylation in mammalian embryos (Allis and Jenuwein, 2016 Cedar and Bergman, 2009). DNA methylation provides a stable and mitotically heritable mode of silencing, which is temporarily erased during gametogenesis and upon fertilization, with re-methylation occurring at the time of implantation (Reik et al., 2001 Smith et al., 2012 Wu et al., 2016). During this time, H3K9me3 mediates gene and transposon repression, and guides the re-establishment of DNA methylation later on (Allis and Jenuwein, 2016 Cedar and Bergman, 2009). As development progresses, pluripotency-associated genes are silenced, while genes involved in alternative cell fates become activated these processes also involve H3K9me3. High-resolution mapping of H3K9me3 in the mouse embryo by ChIP-seq has revealed a finely regulated timing of H3K9me3 establishment at different genomic elements (Wang et al., 2018). H3K9me3 is present at some developmental genes and some long terminal repeats (LTRs) in oocytes and zygotes, but is lost at the two-cell stage. However, globally the two-cell stage is characterized by a stark increase in H3K9me3, which initially accumulates predominantly on LTRs (Wang et al., 2018). Depletion of SetDB1, KAP1/Trim28, Sumo2 and the histone chaperone Chaf1a leads to H3K9me3 loss and upregulation of several LTRs. In addition, many embryos arrest at the blastocyst stage upon knockdown of these factors, highlighting their importance for proper early development (Wang et al., 2018). As development continues into the implantation stage, H3K9me3 also begins to appear at host genes where different lineages acquire distinct H3K9me3 signatures. Typically, in a specific cell type H3K9me3 is deposited at genes that are characteristic of alternative cell fates (Wang et al., 2018). Thus, the H3K9me3 mark appears to suppress lineage-inappropriate gene expression.

The role of SetDB1-mediated TE, and gene repression and cell fate control is also apparent from studies in mouse ESCs. As in early embryos, LTRs in ESCs are marked by H3K9me3. Depletion of KAP1/Trim28, SetDB1/ESET and its co-factor MCAF1/ATF7IP, the KRAB-ZFP Zfp809, Morc2a, Chaf1a/b, Sumo2 and SUMO pathway enzymes leads to pervasive upregulation of multiple (partially overlapping) ERV targets (Cossec et al., 2018 Fukuda et al., 2018 Karimi et al., 2011 Martens et al., 2005 Matsui et al., 2010 Mikkelsen et al., 2007 Rowe et al., 2010 Wolf and Goff, 2007 Yang et al., 2015). In addition, depletion of the H3K9me3 effectors SetDB1, KAP1 and several KRAB-ZFPs from ESCs leads to de-repression of a subset of protein-coding genes (Ecco et al., 2016 Karimi et al., 2011 Rowe et al., 2013 Wolf et al., 2015b). Notably, a significant fraction of genes activated upon SetDB1 or Trim28 depletion reside in proximity to TEs (mostly ERV and LINEs), and many become transcribed from alternative TSSs residing in concomitantly upregulated ERV regions, thereby forming chimeric transcripts (Karimi et al., 2011 Rowe et al., 2013). Furthermore, SetDB1 and H3K9me3 have been reported to occupy promoter regions in ESCs and early embryos, suggesting that they directly target specific host genes (Bilodeau et al., 2009 Karimi et al., 2011 Wang et al., 2018 Yuan et al., 2009). Consistent with a role for H3K9me3-dependent silencing in maintaining cell fate, depletion of silencing factors from ESCs is generally associated with loss of cell identity. For example, depletion of KAP1/Trim28, Chaf1a, SUMO2/3 or the SUMO E2 ligase Ubc9 leads to conversion of the transcription profile of ESCs to a state resembling that of the two-cell embryo, i.e. a two-cell (2C)-like state, suggesting that these factors maintain the ESC state by repressing 2C-specific genes, including the master regulator of the 2C state Dux (Cossec et al., 2018 Ishiuchi et al., 2015 Macfarlan et al., 2012). Elimination of SetDB1 from ESCs also alters their fate, with cells reported to either die or shift to trophoblast-like fate (Bilodeau et al., 2009 Yeap et al., 2009 Yuan et al., 2009). A large fraction of genes repressed by SetDB1/H3K9me3 are developmental regulators (Bilodeau et al., 2009 Karimi et al., 2011 Yuan et al., 2009). Notably, a subset of genes repressed by SetDB1/H3K9me3 in ESCs also encode factors normally expressed in testis and oocytes (Karimi et al., 2011). A recent study suggested that SetDB1 can be directly guided to at least some germline-specific genes in ESCs by the transcription factor MAX (Tatsumi et al., 2018). Moreover, many genes associated with the germline transcriptional program, such as P-granule components and meiosis genes, are also occupied by SUMO in ESCs (Cossec et al., 2018). Together, these data suggest that, in ESCs, H3K9me3-mediated repression involving SetDB1 and SUMO also plays an important role in maintaining cell identity by suppressing alternative fates. Interestingly, some targets of SetDB1/H3K9me3 in ESCs are also marked by H3K27me3 and DNA methylation, indicating several layers of repression for certain genomic targets (Bilodeau et al., 2009 Karimi et al., 2011).

H3K9me3 functions in lineage commitment and cell differentiation

Gene repression through SetDB1-dependent H3K9 methylation is not restricted to ESCs and pre-gastrulation embryos. Despite a prevailing model that TEs in adult somatic tissues of mammals are silenced by DNA methylation, KRAB-ZFP/KAP1/SetDB1-dependent transcriptional repression was reported to control several cell type-specific subsets of ERVs in a range of adult mouse cell types, including embryonic fibroblasts (MEFs), pre-adipocytes, hepatocytes and B-lymphocytes (Collins et al., 2015 Ecco et al., 2016 Fasching et al., 2015 Kato et al., 2018 Wolf et al., 2015b).

Multiple examples highlight a role for H3K9me3 in cell type-specific gene regulation. High resolution analysis of heterochromatin formation in murine cells from different germ layers, and from hepatic and pancreatic lineages revealed that the number of H3K9me3-marked regions in different lineages increases from early developmental stages until gastrulation, although H3K9me3 is subsequently removed as cells progress into specific lineages (Nicetto et al., 2019). Transient deployment of H3K9me3 in germ layer cells is required to repress genes associated with mature cell function, and failure to properly establish this mark leads to expression of lineage-inappropriate genes later on (Nicetto et al., 2019). Silencing by SUV39H1-dependent H3K9me3 and HP1α deposition was shown to be involved in lineage commitment of Th2 lymphocytes by repressing Th1-specific loci (Allan et al., 2012), and in adipogenesis by restricting the expression of master regulatory genes until differentiation is required (Matsumura et al., 2015). H3K9me3-mediated regulation of host genes and TEs by SetDB1 has also been implicated in transcriptome regulation and normal cell fate switches during murine neurogenesis and oligodendrocyte differentiation (Jiang et al., 2017 Liu et al., 2015 Tan et al., 2012). Notably, SetDB1-repressed genes in neuronal tissue are enriched in factors characteristic for other lineages, and particularly in germline-specific genes (Tan et al., 2012). Germline genes are also targets of SetDB1- and SUMO-mediated repression in ESCs (as mentioned above), pointing to a ubiquitous role of this pathway in suppressing germ cell fate. Finally, Hi-C analysis of SetDB1-depleted postnatal mouse forebrain neurons revealed alterations in chromosomal conformation resulting from CTCF binding to cryptic sites normally occupied by H3K9 and DNA methylation (Jiang et al., 2017).

The H3K9me3 mark has also been found to impede cell re-programming (Becker et al., 2016). Studies in human embryonic fibroblasts, for example, identified over 200 H3K9me3-enriched genomic regions, with an average size of 2.2 Mb, that are refractory to binding of the pioneer transcription factors Oct4, Sox2, Klf4 and Myc (OKSM), thereby impeding re-programming to pluripotency (Soufi et al., 2012). Knockdown of the HMTs SUV39H1 and SetDB1, the histone chaperone CAF1 subunits Chaf1a and Chaf1b, Cbx3/HP1γ, Sumo2 and SUMO pathway components, or overexpression of the Jmjd2c demethylase, improve OKSM binding and reprogramming of fibroblasts to induced pluripotent cells in human or murine systems (Borkent et al., 2016 Cheloufi et al., 2015 Chen et al., 2013 Cossec et al., 2018 Onder et al., 2012 Soufi et al., 2012 Sridharan et al., 2013). Similar ‘reprogramming-resistant regions’ (RRRs) marked by H3K9me3 impede epigenetic reprogramming upon somatic cell nuclear transfer, with overexpression of the H3K9 demethylase Kdm4d or simultaneous depletion of the two SUV39 paralogs partially releasing this impediment (Matoba et al., 2014). Notably, a detailed study of the role of SUMO in the reprogramming of MEFs to pluripotency revealed that, in this context, SUMO is required to maintain the activity of fibroblast-specific enhancers (Cossec et al., 2018). This function is in stark contrast to the role of SUMO in ESCs, where it suppresses RRRs and the 2C-like transcriptome, highlighting a context-dependent function of protein SUMOylation (Cossec et al., 2018).

The role of H3K9me3 in somatic tissues in fruit flies is less well understood. dSetDB1/Eggless is the only essential HMT in D. melanogaster. dSetDB1 appears to be responsible for initial deposition of H3K9me3 and HP1 at many regions in the early embryo (Seller et al., 2019), and dSetDB1 mutations are associated with a wide variety of developmental defects and lethality (Brower-Toland et al., 2009 Stabell et al., 2006 Tzeng et al., 2007). As SetDB1-dependent H3K9me3 is present at multiple genomic loci, including nearly the entire chromosome 4 (which contains 79 genes and is enriched in repeats), the severe phenotypes of SetDB1 loss-of-function mutations are likely due to pleiotropic effects. Factors that recruit SetDB1 to its genomic targets in fly somatic tissues have yet to be established. In Drosophila, mutations in RNAi factors, including Piwi, affect PEV and H3K9me3 in somatic tissues not known to have an active piRNA pathway (Gu and Elgin, 2013 Pal-Bhadra et al., 2004). As piRNA/Piwi are maternally loaded into the egg (but not zygotically expressed outside of the gonads), an attractive model is that maternal piRNA/Piwi complexes guide initial heterochromatin establishment in the early embryo, which is later maintained piRNA independently. There are also some H3K9me3-marked genes in regions that do not have local TEs and cannot be targeted by piRNA, pointing to the existence of piRNA-independent targeting mechanisms (Ninova et al., 2019b). DNA-binding proteins that recruit SetDB1 analogous to the vertebrate-specific KRAB-ZFP family have not been identified.

H3K9me3 and gene silencing in germ cells

Germline specification, gonad development and gametogenesis are highly orchestrated processes associated with extensive epigenetic re-programming. As germ cells carry the genetic material to be transmitted to offspring, they must also be well protected from damaging TE activity. Chromatin modification by H3K9me3 plays an essential role in germ cell development and fertility in both vertebrate and invertebrate animals. Most current understanding of transcriptional repression by H3K9me3 in germ cells comes from studies in the male germline of mice, and in the female germline of Drosophila.

In mice, a population of epiblast cells in the post-implantation embryo forms primordial germ cells (PGCs): the precursors of oocytes and spermatozoa. SetDB1 depletion at early stages of development (prior to E6.5 by Sox2Cre cKO and at E9.5 by TnapCre cKO) was shown to repress PGC formation and lead to gonadal hypotrophy in adults (Liu et al., 2014 Mochizuki et al., 2018). During PGC-like cell induction, SetDB1 was suggested to directly repress several transcription factors involved in mesoderm cell fate, thereby maintaining proper cell identity (Mochizuki et al., 2018). In E13.5 PGCs, SetDB1 was shown to control H3K9me3 levels and repress a subset of retrotransposons from the ERV and LINE1 classes, as well as a number of host genes (Liu et al., 2014). As in other systems, many genes deregulated upon SetDB1 loss are not directly marked by H3K9me3 but reside in the proximity of or initiate their transcription from within TEs (Liu et al., 2014). The factors that guide H3K9 methylation by SetDB1 in early PGCs are not known. Metazoan germline cells typically possess an active piRNA pathway. However, Miwi2, the only nuclear Piwi protein in mice, is not expressed until E14.5-15.5 (Aravin et al., 2008), thus H3K9me3 deposition before this stage is likely piRNA independent. It is possible that, as in other tissues, TEs in early germ cells are repressed by KRAB-ZFPs, but this hypothesis needs to be addressed.

In addition to functioning in PGCs and testis, SetDB1 has been shown to regulate the expression of host genes, several TEs and associated chimeric transcripts in mouse oocytes (Eymery et al., 2016 Kim et al., 2016). While mammalian oocytes express Piwi proteins and piRNAs, the mechanisms of H3K9me3 establishment at different genomic targets in this system has not been comprehensively characterized.

In Drosophila, H3K9me3-mediated silencing is best understood in the ovary. Of the two main H3K9 HMTs that induce trimethylation in Drosophila, SetDB1/Eggless is required throughout the entire course of oogenesis, from germ cell differentiation to egg maturation, as well as for the somatic follicular cells that support the ovary, while Su(var)3-9 is not essential for fertility (Clough et al., 2007 Clough et al., 2014). Functionally, SetDB1/Eggless acts at multiple levels, including the control of TE expression by the piRNA pathway and repression of lineage-specific genes. Unlike Su(var)3-9 (Sienski et al., 2015), SetDB1 is involved in piRNA-dependent TE repression not only by being part of the piRNA-mediated transcriptional silencing pathways but also by regulating piRNA production. In D. melanogaster, primary piRNAs are generated from discrete genomic loci termed piRNA clusters (Brennecke et al., 2007). Most piRNA clusters in germ cells are characterized by a unique epigenetic landscape consisting of H3K9me3, the germline-specific HP1 variant Rhino/HP1d (a Drosophila-specific HP1 homolog) and several other factors that are required for their transcription and piRNA production (Andersen et al., 2017 Chen et al., 2016 Mohn et al., 2014 Rangan et al., 2011). In the nucleus, piRNA-loaded Piwi proteins recognize nascent transcripts of active TEs and induce local H3K9 trimethylation and co-transactional silencing (Klenov et al., 2011 LeThomas et al., 2013 Rozhkov et al., 2013 Sienski et al., 2012). SetDB1/Eggless depletion leads to H3K9me3 loss from TE targets and loss of piRNAs (Rangan et al., 2011). While loss of H3K9me3 at TE targets is probably partly due to loss of piRNA guides, several lines of evidence show that SetDB1 is also directly involved in H3K9me3 deposition downstream of the piRNA/Piwi complex. Piwi is not known to interact with any HMTs. However, two of Piwi's interacting partners, Panoramix (Panx)/Silencio and the SUMO E3 ligase Su(var)2-10, induce H3K9me3 deposition when recruited to chromatin in a process that is dependent on SetDB1 and its conserved co-factor Wde (ATF7IP/MCAF1 in mammals) (Ninova et al., 2019a preprint Sienski et al., 2015 Yu et al., 2015). Furthermore, SetDB1/Wde recruitment requires SUMO and the SUMO E3 ligase activity of Su(var)2-10 (Ninova et al., 2019a preprint). Collectively, these findings lead to a model in which Su(var)2-10 interacts with Piwi/Arx/Panx and acts to induce SUMO-dependent recruitment of Wde/SetDB1, which in turn deposits H3K9me3 at piRNA targets (Ninova et al., 2019a preprint) (Fig. 3B).

As in mammalian systems, epigenetic silencing of TEs affects the host transcriptome of Drosophila germ cells. For example, H3K9me3 loss is associated with activation of cryptic promoters within TE sequences, the appearance of chimeric or truncated transcripts and mis-regulation of canonical gene isoforms (Ninova et al., 2019b). Finally, even though the piRNA pathway is the only known mode of H3K9me3 deposition in Drosophila ovaries, a recent ChIP-seq study revealed a number of discrete H3K9me3 peaks at euchromatic genes that are conserved, show no evidence of TE insertions or targeting by piRNAs, and do not lose H3K9me3 upon Piwi depletion, i.e. are likely piRNA-independent (Ninova et al., 2019b). About 20% of these H3K9me3-marked genes become upregulated upon knockdown of the SUMO ligase Su(var)2-10, suggesting that they are regulated in a SUMO-dependent manner and possibly through SetDB1. Notably, this set primarily includes genes characteristic of other tissues such as the testis or the central nervous system (Ninova et al., 2019b). It was recently demonstrated that the H3K9me3 effectors SetDB1, Wde and HP1a are required to confer transcriptional repression of male germline fate in the ovary (Smolko et al., 2018). Among other targets, SetDB1/Wde-dependent H3K9me3 suppresses the male-specific isoform of the master regulator of sex identity phf7 (Smolko et al., 2018). Thus, in addition to its role in constitutive heterochromatin and TE repression, epigenetic regulation by H3K9me3 in the female germline appears to grant tissue-specific gene repression to secure female germ cell identity (Ninova et al., 2019b Smolko et al., 2018). The presence of discrete and TE-independent H3K9me3 peaks in otherwise euchromatic regions in female germ cells suggests the existence of a piRNA-independent mode of SetDB1 recruitment, and a regulatory mechanism that restricts H3K9me3 spreading in this genomic context.

Interestingly, a recent study in Drosophila showed a role for the conserved factor L(3)mbt in lineage-inappropriate gene repression in the female germline and soma (Coux et al., 2018). The mammalian L3MBTL2 homolog (involved in PRC1.6) is also required for the repression of germline-specific genes in mouse ESCs (Maeda et al., 2013 Stielow et al., 2018 Tatsumi et al., 2018). In the future, it would be worthwhile comparing targets of SetDB1/H3K9me3 and other silencing complexes, and investigating any potential cooperation between them.

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Zinc finger proteins orchestrate active gene silencing during embryonic stem cell differentiation. / Kwak, Sojung Kim, Tae Wan Kang, Byung Hee Kim, Jae Hwan Lee, Jang Seok Lee, Han Teo Hwang, In Young Shin, Jihoon Lee, Jong Hyuk Cho, Eun Jung Youn, Hong Duk .

In: Nucleic Acids Research , Vol. 46, No. 13, 27.07.2018, p. 6592-6607.

Research output : Contribution to journal › Article › peer-review

T1 - Zinc finger proteins orchestrate active gene silencing during embryonic stem cell differentiation

N1 - Publisher Copyright: © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

N2 - Transcription factors and chromatin remodeling proteins control the transcriptional variability for ESC lineage commitment. During ESC differentiation, chromatin modifiers are recruited to the regulatory regions by transcription factors, thereby activating the lineage-specific genes or silencing the transcription of active ESC genes. However, the underlying mechanisms that link transcription factors to exit from pluripotency are yet to be identified. In this study, we show that the Ctbp2-interacting zinc finger proteins, Zfp217 and Zfp516, function as linkers for the chromatin regulators during ESC differentiation. CRISPR-Cas9-mediated knock-outs of both Zfp217 and Zfp516 in ESCs prevent the exit from pluripotency. Both zinc finger proteins regulate the Ctbp2-mediated recruitment of the NuRD complex and polycomb repressive complex 2 (PRC2) to active ESC genes, subsequently switching the H3K27ac to H3K27me3 during ESC differentiation for active gene silencing. We therefore suggest that some zinc finger proteins orchestrate to control the concise epigenetic states on active ESC genes during differentiation, resulting in natural lineage commitment.

AB - Transcription factors and chromatin remodeling proteins control the transcriptional variability for ESC lineage commitment. During ESC differentiation, chromatin modifiers are recruited to the regulatory regions by transcription factors, thereby activating the lineage-specific genes or silencing the transcription of active ESC genes. However, the underlying mechanisms that link transcription factors to exit from pluripotency are yet to be identified. In this study, we show that the Ctbp2-interacting zinc finger proteins, Zfp217 and Zfp516, function as linkers for the chromatin regulators during ESC differentiation. CRISPR-Cas9-mediated knock-outs of both Zfp217 and Zfp516 in ESCs prevent the exit from pluripotency. Both zinc finger proteins regulate the Ctbp2-mediated recruitment of the NuRD complex and polycomb repressive complex 2 (PRC2) to active ESC genes, subsequently switching the H3K27ac to H3K27me3 during ESC differentiation for active gene silencing. We therefore suggest that some zinc finger proteins orchestrate to control the concise epigenetic states on active ESC genes during differentiation, resulting in natural lineage commitment.


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Keywords : Hypoxia-inducible factor-1α, WNT7a, myogenesis, hypertrophy, Prolyl-hydroxylases, FG-4592

Citation: Cirillo F, Resmini G, Angelino E, Ferrara M, Tarantino A, Piccoli M, Rota P, Ghiroldi A, Monasky MM, Ciconte G, Pappone C, Graziani A and Anastasia L (2020) HIF-1α Directly Controls WNT7A Expression During Myogenesis. Front. Cell Dev. Biol. 8:593508. doi: 10.3389/fcell.2020.593508

Received: 10 August 2020 Accepted: 20 October 2020
Published: 11 November 2020.

Susan Tsivitse Arthur, University of North Carolina at Charlotte, United States

Eoin P. Cummins, University College Dublin, Ireland
Sivareddy Kotla, University of Texas MD Anderson Cancer Center, United States
Colin E. Evans, Northwestern University, United States

Copyright © 2020 Cirillo, Resmini, Angelino, Ferrara, Tarantino, Piccoli, Rota, Ghiroldi, Monasky, Ciconte, Pappone, Graziani and Anastasia. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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