An Overview of Novel Transcription Factors Involved in Spermatogonial Stem Cells

Abstract

A unique subset of spermatogonial stem cells (SSCs) initiates and maintains spermatogenesis. These SSCs have unique morphological traits attached to the seminiferous tubules basement membrane. They provide the groundwork for a healthy stem cell system in the testis, which is essential for spermatogenesis and other reproductive functions. The fascinating proteins known as transcription factors (TFs) have a great deal of control over gene expression in all living things. Some TFs are essential to the coordination of the complex dance known as spermatogenesis. Certain mutations in TFs may lead to the disorder of spermatogenesis. Distinguishing these TFs will be helpful to understand spermatogenesis and to locate possible therapeutic targets. In this chapter, we will review the recently identified TFs including E4F1, FoxP4, A-MYB, TCFL5, and TCF3 that play a role in SSCs. Enrich Shiny gene ontology and Cytoscape tools were used to predict the molecular connections and functional characteristics of proteins in order to find the key pathways. Our bioinformatic analysis will help us to understand these new and important connections between the TFs and the remaining gene expression in the protein network.

1. Introduction

In the male reproductive system, spermatogenesis is one of the most important processes. Spermatogonia stem cells (SSCs) play the most fundamental role in this system [1]. The process of spermatogenesis involves multiple steps involving SSCs that simultaneously undergo self-renewal and differentiation and then undergo sequential cell-fate transitions between mitosis and meiosis to produce highly specialized sperms [2]. The process of spermatogenesis also involves spermiogenesis (a spermatogenesis is the metamorphosis of spermatogonia into spermatozoa and includes all functional and structural changes that occur throughout this process) [3, 4]. During the course of spermatogenesis, cells undergo a number of stages where they progress from spermatogonia to mature sperm [5]. In spermatogenesis, genetic information is passed on to the next generation [6]. When it comes to spermatogenesis, it is very important that proteins are expressed in the proper order and location at crucial stages of the process [7]. Biological evolution is based on the variable expression of genetic information, and an organism’s ability to adapt to changes in its environment is largely dependent on the control of gene expression. Gene expression levels, location, and timing are all strictly regulated. Gene regulation is a multifaceted and intricate process that primarily operates at the transcriptional level. Transcription factors bind to certain DNA sequences to modulate chromatin shape and target gene transcription through intricate networks of interacting factors [8]. SSCs are thought to be a promising substitute for injured or compromised spermatogenesis in order to regenerate it [9].

While a number of spermatogonial stem cell regulators have been well studied in rodents, the regulatory mechanisms governing the self-renewal and differentiation of human SSCs remain incompletely understood [10]. The transcription factors Promyelocytic leukemia zinc finger protein (Plzf) and TATA-box binding protein associated factor 4b (Taf4b) have previously been linked to the regulation of SSC functions; these molecules are a part of a complex gene network that determines the fate of SSCs [9]. There are a number of important gene expression regulators in spermatogenesis that have been found, especially in spermiogenesis. For instance, alternate splicing of the cAMP responsive element modulator (CREM) transcripts in pachytene SCs (pacSCs) and delayed translation in round spermatids (rSTs) result in the expression of CREM-τ, a transcription activator that is exclusively produced in rSTs. Spermatogenesis is stopped in mice with CREM gene knockouts (KO) at the early rST stage. Numerous genes involved in a variety of biological activities have their expression directly or indirectly regulated by CREM-τ. These characteristics have led to the recognition of CREM-τ as a crucial regulator of spermiogenesis. Transcription factors (TFs) like TRF2, TAF7L, and RFX2 and RNA-binding proteins (RBPs) like TPAP, MIWI, RNF17, BOULE/BOLL, and GRTH/DDX25 are examples of similar regulators [11]. Whereas a number of spermatogonial stem cell regulators have been well studied in rodents, the regulatory mechanisms governing the self-renewal and differentiation of human SSCs remain incompletely understood [6, 7, 10, 12, 13, 14, 15, 16, 17].

We will go over the newly discovered TFs involved in SSCs in this book chapter. Furthermore, the bioinformatic analyses performed resulted in the comprehension and identification of novel and significant associations between these transcription factors and the remaining genes within the protein network.

2. Some novel transcription factors involved in spermatogonial stem cell proliferation and differentiation

2.1 FOXP4

The “Forkhead” motif-bearing Forkhead box (Fox) family of transcription factors is an essential component of human health and sickness that has been preserved throughout evolution. Apoptosis, cell differentiation, aging, embryonic development, glucose and lipid metabolism, and immunological control are all impacted by genes belonging to the Fox family [18]. It has been recently established that the FOXP subfamily is a part of the Fox gene family. In addition to having a forkhead domain, members of the FOXP subfamily also have a zinc finger domain and a leucine zipper motif [19]. Tucked down on human chromosome 6p21.1, forkhead box P4 (FOXP4) is a member of the FOXP subfamily that codes for a 680 amino acid protein [20].

According to recent research, FOXP4 may be involved in the oncogenesis of several tissues, including hepatocellular carcinoma, prostate cancer, and non-small-cell lung cancer. In addition to being well-known for controlling tissue growth or cell differentiation throughout life, the FOXP family (FOXP1-4) is also important for oncogenesis, cell-cycle regulation, and embryonic development. Among them, it has been documented that FOXP4 controls the differentiation and regeneration of lung secretory epithelial cells [21].

Luo et al. examined human testis single-cell sequencing data and discovered that the expression of forkhead box P4 (FOXP4) progressively increased as SSCs developed [10]. Subsequent examination of FOXP4’s expression patterns in human testicular tissues showed that it identifies spermatogonia with stem cell potential in a distinct subgroup. In human SSC lines, conditional FOXP4 silencing greatly increased apoptosis and inhibited SSC growth. The expression of FOXP4 was significantly reduced in areas where spermatogenesis was dysregulated. These results suggest that FOXP4 plays a role in human SSC proliferation, which will clarify the processes governing the decisions made about the fate of human SSCs [10].

2.1.1 Analyzing FOXP4 in gene network and bioinformatic tools

The PPI network was visualized using the Cytoscape software version 3.10.1 (https://cytoscape.org/) as shown in Figure 1A. BCL6, CTBP1, DHDDS, EZH2, FOXP1, FOXP1-2, FOXP2, FOXP3, GATAD2B, HAND1, HAND2, LCOR, NFATC2, PRR20D, PRR20E, SATB1, SRPX2, TBR1, TINCR, and ZNF609 are the associated genes.

Figure 1.

Bioinformatic analysis of FOXP4: (A) protein–protein interaction (B) table of top 10 significant p-values for GO biological process (C) table of top 10 significant p-values for GO molecular function (D) KEGG pathway.

Terms related to the biological process (BP) and molecular function (MF) were revealed using functional enrichment analysis (https://maayanlab.cloud/Enrichr/). Moreover, the biological pathway mediated by related TRANSCRIPTION FACTOR genes was found using the ShinyGO tool (http://bioinformatics.sdstate.edu/go/). SATB1, CTBP1, NFATC2, TBR1, FOXP4, FOXP3, FOXP2, GATAD2B, FOXP1, ZNF609, BCL6, HAND2, HAND1, LCOR, and EZH2 are involved in the regulation of DNA-templated transcription (GO:0006355) and transcription by RNA polymerase II (GO:0006357).

The following associated molecular function terms were also looked into: regulation of DNA-templated transcription (GO:0006355), regulation of transcription by RNA polymerase II (GO:0006357) for the proteins (SATB1, CTBP1, NFATC2, TBR1, FOXP4, FOXP3, FOXP2, GATAD2B, FOXP1, ZNF609, BCL6, HAND2, HAND1, LCOR, and EZH2).

2.2 A-MYB

Nuclear DNA-binding proteins known as myoblastosis (MYB) proteins function as transcriptional transactivators of several genes. Target genes that control cell cycle development, differentiation, and apoptosis are bound by them and transcriptionally stimulated. Mammals possess three distinct MYB paralogs: A-MYB, B-MYB, and C-MYB (correspondingly, MYBL1, MYBL2, and MYB in mice). Spermatocytes following meiotic entry have significant levels of MYBL1 transcription. Histological analysis indicates that the male infertility is caused by meiosis arrest at around the pachytene stage. Thus, it is plausible that MYBL1 functions as a crucial regulator of spermatocyte meiosis [22].

According to the study, a little alteration in transcription factor regulation upstream might have a cascade impact downstream in a regulatory network, resulting in hybrid male sterility. The relationship between the key transcription factor and hybrid male sterility was investigated using RNA sequencing, assay for transposase-accessible chromatin, and high-throughput sequencing analyses. It was discovered that the widespread misexpression in dzo was linked to spermatogenesis-related genes as well as somatic or progenitor genes. The male meiosis-specific master transcription factor MYBL1 motif was shown to be abundant on the promoters of downregulated pachytene spermatocyte genes in dzo, according to the study of transcription factor-binding motif enrichment. The MYBL1-binding motif’s target genes were shown to be considerably downregulated in the dzo testis and linked to genes unique to meiosis [23].

Promoters, super enhancers, and endogenous retroviral enhancers that stimulate meiotic gene transcription are bound by A-MYB. A-MYB directly binds the promoters of less than 25% of meiotic genes, indicating that one or more additional transcription factors act downstream or in parallel to A-MYB to regulate meiotic gene transcription, even though A-MYB-mutant germ cells fail to express meiotic genes and arrest early in meiosis I [24].

2.2.1 The outcomes of bioinformatic analyses on MYBL-1

The protein–protein interaction network (Figure 2A) indicated the related genes including BCL7A, CREBBP, E2F2, E2F4, E2F5, FOXM1, LIN37, LIN52, LIN54, LIN9, LMO2, MYBL2, NCL, NFIB, RBBP4, RBL1, RBL2, TESMIN, TFDP1, and TFDP2.

Figure 2.

Bioinformatic analysis of MYBL1: (A) protein–protein interaction (B) table of top 10 significant p-values for GO biological process (C) table of top 10 significant p-values for GO molecular function (D) KEGG pathway.

CREBBP, LIN37, LMO2, LIN9, FOXM1, TFDP1, TFDP2, BCL7A, NFIB, E2F2, MYBL2, E2F4, E2F5, and MYBL1 all participated in the two biological processes (regulation of transcription by RNA polymerase II (GO:0006357) and regulation of DNA-templated transcription (GO:0006355)). Furthermore, the linked molecular activities of this gene network include RNA Polymerase II transcription regulatory region sequence-specific DNA binding (GO:0000977), DNA-binding transcription activator activity, and RNA polymerase II-specific (GO:0001228).

2.3 TCFL5

Transcription factor-like 5 (TCFL5) stimulates the transcription of genes involved in meiosis, mRNA turnover, the generation of miR-34/449, meiotic exit, and spermiogenesis [24]. TCFL5 is found in the middle and late spermatocytes, beginning with stage V and progressing to the round spermatid at stage I, and has nuclear and cytoplasmic expression [25].

MYBL1 activates TCFL5 expression; in mice with defective MYBL1 testes, no TCFL5 protein is seen. Cleavage under targets and release using nuclease (CUT&RUN) for MYBL1 from FACS-purified male germ cells found MYBL1 occupancy around the TCFL5 gene promoter, supporting the same line of evidence. It is interesting to note that TCFL5 mutant testes had lower MYBL1 mRNA and protein levels. Interestingly, CUT&RUN for TCFL5 also recorded TCFL5 occupancy on the Mybl1 promoter, demonstrating that TCFL5 and MYBL1 positively feedback on each other’s transcription [26]. Through high-throughput sequencing of TCFL5 ChIP-DNA and eCLIP-RNA, the study discovered that TCFL5 controls a group of genes involved in the formation of male germ cells both transcriptionally and posttranscriptionally. Additionally, the protein interacts with FXR1, a known RNA-binding protein that may be involved in coordinating TCFL5’s transition and nucleus localization [25].

Galán et al. created two TCFL5 deletion mice models in order to study the function of TCFL5 in spermatogenesis [27]. Male mice that were part of the whole TCFL5-deficient mice paradigm had infertility due to Sox2 promoter-controlled CRE recombinase. This model’s phenotypic investigation showed that TCFL5 is required for the progression of meiosis. Furthermore, the tamoxifen-conditional knockout mouse demonstrated that TCFL5, in addition to the previously mentioned functions, plays a role in spermiogenesis. According to gene expression research, the transcription factor TCFL5 may be able to regulate key spermatogenesis genes that are involved in mitosis, meiosis, and spermiogenesis. These findings highlight the critical function of TCFL5 in the advancement of spermatogenesis, which is necessary for the resolution of meiosis and the maturation of spermatids [27]. A further research conducted in 2023 on conservation in rhesus macaques indicates that TCFL5 is essential for placental animals’ meiosis and spermiogenesis [24].

2.4 Gene network analysis related to TCFL5 and MYBL1

We chose to input these two genes into the program simultaneously in order to find any associated proteins because of the connection between them and their regulatory pathway. As in Figure 3A, ARFGEF1, BCL7A, GPR183, IL21, KIAA1549, LIN37, LIN52, LIN54, LIN9, LMO2, LRMP, MORN3, MOV10L1, NFIB, PDCD2, PPFIBP2, RBBP4, TMOD3, and ZFX were detected in the protein network. BCL7A, RBBP4, PDCD2, and TCFL5 are involved in the regulation of cell differentiation (GO:0045595). LIN37, BCL7A, RBBP4, NFIB, LMO2, LIN9, ZFX, MYBL1, and TCFL5 take place in the regulation of transcription by RNA polymerase II (GO:0006357). Moreover, MYBL1 and MOV10L1 can take part in male meiosis I (GO:0007141). Functional enrichment analysis demonstrated the biological process (BP) terms cis-regulatory region sequence-specific DNA binding (GO:0000987) and RNA polymerase II cis-regulatory region sequence-specific DNA binding (GO:0000978) ([NFIB, ZFX, MYBL1, TCFL5).

Figure 3.

Bioinformatic analysis of TCFL5: (A) protein–protein interaction (B) table of top 10 significant p-values for GO biological process (C) table of top 10 significant p-values for GO molecular function (D) KEGG pathway.

2.5 TCF3

The E protein E2A, a member of the basic helix–loop–helix (b-HLH) transcription factor family, is encoded by transcription factor 3 (TCF3). Two unique isoforms, E47 and E12, are produced by alternative splicing; they only vary in exon 18 pertaining to the b-HLH domain [28]. To carry out their tissue- or cell type-specific tasks, TCF3 proteins combine with other b-HLH proteins to create homodimers or heterodimers. The TCF3 protein is dysregulated in a number of malignancies, including lymphoma, pancreatic cancer, breast cancer, colorectal cancer, and prostate cancer. It was first shown to play especially significant functions during lymphocyte development. Numerous investigations have demonstrated that the TCF3 protein functions as an oncoprotein or a tumor suppressor [29].

The co-occupancy of TCF3 with Oct4, Sox2, and Nanog on embryonic stem cell (ESC) chromatin indicated that TCF3 has been suggested to play an integral role in a poorly understood mechanism underlying wingless-related integration site-dependent stimulation of mouse ESC self-renewal of mouse ESCs [30].

According to a recent study, GFRA1-positive spermatogonia were the main source of TCF3 expression, and TCF3 expression was upregulated by the epidermal growth factor (EGF). Interestingly, TCF3 inhibited human SSC apoptosis while promoting SSC proliferation and DNA synthesis. TCF3 protein controlled the transcription of many genes, including WNT2B, TGFB3, CCN4, MEGF6, and PODXL, according to RNA sequencing and chromatin immunoprecipitation (ChIP) experiments. PODXL silencing, on the other hand, reduced the stem cell activity of SSCs. Furthermore, in patients with spermatogenesis failure, the amount of TCF3 protein was significantly lower than in those with obstructive azoospermia who had normal spermatogenesis. Altogether, our findings suggested that TCF3 regulates PODXL-mediated human SSC proliferation and apoptosis [31].

2.5.1 Bioinformatics analysis of TCF3

The gene was uploaded in Cytoscape software, and the related proteins are demonstrated in Figure 4A. (TCF3, CBFA2T3, CBFB, GATA1, GFI1B, ID1, ID2, LDB1, LDB2, LMO1, LMO2, LYL1, MYOD1, RUNX1, SSBP2, SSBP3, TAL1, TAL2, TCF12, TLX1, and ZFPM1). The related biological processes after enrichment were: DNA-binding transcription factor binding (GO:0140297), RNA polymerase II-specific DNA-binding transcription factor binding (GO:0061629), cis-regulatory region sequence-specific DNA binding (GO:0000987), RNA polymerase II cis-regulatory region sequence-specific DNA binding (GO:0000978), RNA polymerase II transcription regulatory region sequence-specific DNA binding (GO:0000977), and DNA-binding transcription activator activity, RNA polymerase II-specific (GO:0001228).

Figure 4.

Bioinformatic analysis of TCF3: (A) protein–protein interaction (B) table of top 10 significant p-values for GO biological process (C) table of top 10 significant p-values for GO molecular function (D) KEGG pathway.

Moreover, the molecular functions of the gene set were: regulation of transcription by RNA polymerase II (GO:0006357), positive regulation of DNA-templated transcription (GO:0045893), regulation of DNA-templated transcription (GO:0006355), positive regulation of transcription by RNA polymerase II (GO:0045944), negative regulation of DNA-templated transcription (GO:0045892), and negative regulation of transcription by RNA polymerase II (GO:0000122).

2.6 E4F1

The human genome’s 16p13.3 region contains the E4F transcription factor 1 (E4F1) gene, which codes for a zinc finger (ZF) protein belonging to the GLI-Kruppel family. This protein is widely acknowledged as a transcription factor and is produced in both humans and mice [32]. The E4F1 protein was initially discovered as a transcriptional regulator of the E4 viral gene, which is hijacked during adenoviral infection by the viral oncoprotein E1A [33].

As a multifunctional transcription factor, E4F1 plays an essential role in cell fate decisions and interacts with Retinoblastoma protein (pRB), a regulator of Sertoli cells [34]. Due to the mortality of the preimplantation embryos in E4F1 knockout mice, the E4F1 is thought as a necessary gene for early embryonic development [32]. A study that used a mouse model with a conditional knockout of the E4F1 gene showed that E4F1 deletion affected Sertoli cell proliferation, which in turn increased germ cell death and decreased testis size and fertility in order to avoid this embryonic mortality [34].

Yan et al. examined undifferentiated spermatogonia’s chromatin accessibility at the single-cell level, and they discovered 37 positive TF regulators that could play a part in determining the fates of SSCs [35]. The whole undifferentiated spermatogonial pool gradually disappears when the transcription factor E4F1, which is expressed in spermatogonia, is conditionally deleted in mouse germ cells. E4F1 functions as a critical modulator of mitochondrial activity, as demonstrated by single-cell RNA-seq study of normal and E4f1-deficient spermatogonia. Ndufs5, Cox7a2, Cox6c, and Dnajc19 are among the genes whose promotor E4F1 binds to and encodes components of the mitochondrial respiratory chain. Undifferentiated spermatogonia were progressively lost as a result of cell cycle arrest and increased apoptosis brought on by aberrant mitochondrial morphology and abnormalities in fatty acid metabolism induced by loss of E4F1 activity. P53 deletion in E4F1-deficient germ cells did not correct the abnormalities in SSC maintenance; rather, it merely momentarily stopped spermatogonial loss [35].

2.6.1 Bioinformatic analysis of E4F1

The network was ultimately extended once this gene was added to the Cytoscape program.

This gene was observed in connection with BMI1, BRAT1, CBX2. CBX4. CBX6, CBX7, CDKN2A, CITED2, COMMD3-BMI1, FAAP100, LRRC14, PHC1, PHC2, RASSF1, SCMH1, TELO2, TP53, WDR24, ZBTB17, and ZGPAT (Figure 5A).

Figure 5.

Bioinformatic analysis of E4F1: (A) protein–protein interaction (B) table of top 10 significant p-values for GO biological process (C) table of top 10 significant p-values for GO molecular function (D) KEGG pathway.

As we are able to perceive, E4F1 with TP53, ZBTB17, and CITED2 are involved in negative regulation of DNA-templated transcription (GO:0045892), regulation of cell cycle (GO:0051726), negative regulation of transcription by RNA polymerase II (GO:0000122), and regulation of transcription by RNA polymerase II (GO:0006357).

Also, the molecular function terms including: RNA polymerase II-specific DNA-binding transcription factor binding (GO:0061629) for CDKN2A, E4F1 and TP53, DNA-binding transcription repressor activity, RNA polymerase II-specific (GO:0001227) for ZGPAT, E4F1 and TP53, DNA-binding transcription factor binding (GO:0140297) and DNA-binding transcription activator activity, RNA polymerase II-specific (GO:0001228) for ZBTB17, E4F1 and TP53. KEGG pathway shows that these genes can be involved in signaling pathways regulating pluripotent stem cells.

Our attention was drawn to Cbp/P300-Interacting Transactivator with Glu/Asp-Rich Carboxy-Terminal Domain 2 (CITED2), a transcription factor that may be involved in the process of spermatogenesis, by further gene network search and analysis. We decided to identify this gene for further research and to elucidate its function in spermatogenesis after reviewing the literature.

2.7 CITED2

CITED2 (a key member of the CITED family), a transcription cofactor, engages with various transcription factors and cofactors, playing crucial roles in fundamental cellular processes such as proliferation, apoptosis, differentiation, migration, and autophagy. Its interacting partners, including LIM homeobox 2, transcription factor AP-2, SMAD2/3, peroxisome proliferator-activated receptor γ, estrogen receptor, MYC, Nucleolin, and p300/CBP, actively regulate downstream gene expression, contributing significantly to the mentioned cellular processes. Recent findings have underscored the indispensable function of CITED2 in both embryonic and adult tissue stem cells, encompassing hematopoietic stem cells and tendon-derived stem/progenitor cells [36, 37]. In the study conducted by Buaas et al. in 2009, it was determined that the transcription cofactor CITED2 plays a crucial role in the processes of sex determination and the early development of gonads [38]. According to the findings of the study conducted by Fang et al. it is proposed that the measurement of CITED2 protein expression in cumulus cells can be a valuable tool for assessing embryo quality and predicting the probability of a successful pregnancy, particularly within the specific context of an in vitro fertilization (IVF) cycle. This implies that monitoring the levels of CITED2 in cumulus cells may offer clinicians and researchers a reliable indicator to evaluate the viability of embryos and anticipate the likelihood of a positive pregnancy outcome during IVF procedures [39].

2.7.1 Bioinformatic analysis of CITED2

Similar to the previous genes, bioinformatic analyses were conducted for this gene, and the following results were obtained. With regard to Figure 6A, CITED2 is in interaction with ARNT, ARNT2, ARNTL, CITED1, CITED4, CREBBP, CRTC2, EP300, EPAS1, FOXO1, FOXO3, HIF1A, HIF3A, KCTD1, LHX2, TFAP2A, TFAP2B, TFAP2C, TFAP2D, and TFAP2E.

Figure 6.

Bioinformatic analysis of CITED2: (A) protein–protein interaction (B) table of top 10 significant p-values for GO biological process (C) table of top 10 significant p-values for GO molecular function (D) KEGG pathway.

TFAP2A, TFAP2B, ARNT2, TFAP2C, CREBBP, CRTC2, TFAP2D, TFAP2E, CITED1, EPAS1, CITED2, ARNT, HIF3A, FOXO3, HIF1A, FOXO1, LHX2, and EP300 are involved in the regulation of DNA-templated transcription (GO:0006355) and transcription by RNA polymerase II (GO:0006357).

3. Conclusion

Spermatogenesis is an intricate process involving numerous gene regulations and transcription factors. Spermatogonial stem cells (SSCs) play a pivotal role in initiating and sustaining spermatogenesis. Transcription factors, by regulating gene expression, activate SSCs, leading to the formation of sperm. These recent discoveries have contributed to a better understanding of spermatogenesis, enabling us to anticipate molecular connections and functional characteristics of proteins involved in SSC activation for sperm formation. This book chapter highlights the crucial role of transcription factors in the intricate regulation of gene expression in SSCs. Our bioinformatics research indicates that the CITED2 gene could play a valuable role in spermatogenesis. Existing studies suggest the potential significance and essential nature of this protein. Nevertheless, further research is necessary to validate this claim. A comprehensive analysis of current research indicates that these transcription factors might serve as biomarkers for gene therapy and other treatments related to male infertility.

Acknowledgments

This study was funded by Center for International Scientific Studies and Collaboration (CISSC) and Amol University of Special Modern Technologies.

Conflict of interest

The authors declare no conflict of interest.

Abbreviations

SSCs	spermatogonia stem cells
TFs	transcription factors
E4F1	E4F transcription factor 1
FOXP4	Forkhead box protein P4
A-MYB	MYB Proto-Oncogene Like 1
TCFL5	transcription factor like 5
TCF3	transcription factor 3
CITED2	Cbp/P300-Interacting Transactivator with Glu/Asp-Rich Carboxy-Terminal Domain 2
ChIP	chromatin immunoprecipitation
RSTs	round spermatids
Plzf	promyelocytic leukemia zinc finger protein