Chromodomain Protein Research Articles

Abstract 921: Cdyl2 is a chromodomain protein involved in the maintenance of pluripotency of stem cells

Abstract Reader proteins of histone modifications are required to translate the information of histone marks into the cellular phenotypes, including pluripotency and malignancy. For example, a reader protein BRD4 has its target specificity and becomes a target of cancer therapy. Here, we aimed to identify reader proteins involved in the pluripotency of stem cells. Using the UCSC database, 11 genes coding chromo- or PHD-domain proteins were isolated as actively transcribed in mouse ES cells (ESCs). Among the 11 genes, Cdyl2, chromodomain protein Y-like2, was found to be down-regulated upon ESC differentiation using RT-qPCR. Immunofluorescence and ChIP assay revealed that Cdyl2 recognized H3K27me3 of the specific regions, including differentiation-associated genes. Cdyl2-knock-out ESCs could not survive after differentiation induced by LIF removal or retinoic acid because of the induction of apoptosis, indicating that Cdyl2 is important for normal differentiation of ESCs. ESC expressing exogenous Cdyl2 showed incomplete down-regulation of Oct-4 and Nanog by retinoic acid, and generated teratomas with an abnormal composition of three germ layers, showing perturbation of differentiation ability. Pathologically, aberrant expression of human CDYL2 was observed in breast cancer cell lines and primary breast cancers. Two breast cancer cell lines (MDA-MB-231 and MDA-MB-468) expressing exogenous CDYL2 showed enhanced attachment ability along with the up-regulation of integrin alpha-6 and integrin beta-1. The population of cancer stem cells, defined as ALDH positive cells, were increased in CDYL2-expressing MDA-MB-468 cells, showing that human CDYL2 is involved in proliferation of cancer stem cells. From these data, we conclude that, between self-renewal and differentiation of stem cells, mouse and human Cdyl2/CDYL2 is necessary for the implementation of the initial step of differentiation. Citation Format: Naoko Hattori, Kana Kimura, Jumpei Taguchi, Toshio Imai, Yasuhiro Yamada, Toshikazu Ushijima. Cdyl2 is a chromodomain protein involved in the maintenance of pluripotency of stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 921. doi:10.1158/1538-7445.AM2017-921

The SERTAD protein Taranis plays a role in Polycomb-mediated gene repression.

The Polycomb group (PcG) proteins have been implicated in epigenetic transcriptional repression in development, stem cell maintenance and in cancer. The chromodomain protein Polycomb (Pc) is a key member of the PcG. Pc binds to the histone mark, trimethylated histone 3 lysine 27 (H3K27me3), to initiate transcriptional repression. How PcG proteins are recruited to target loci is not fully understood. Here we show that the Drosophila SERTA domain protein Taranis (Tara) is involved in transcriptional regulation of Pc target genes. Embryos lacking Tara exhibit a partial homeotic transformation of cuticular the segments, a phenotype associated with the loss of Pc function. Moreover, Drosophila embryos homozygous for a tara hypomorphic allele also misexpress engrailed, a Pc-regulated gene, and this phenotype is associated with the loss of Pc binding to the cis response element in the engrailed enhancer. In relation to that, Pc recruitment is reduced on the salivary gland polytene chromosomes and specifically at the engrailed locus. These results suggest that Tara might be required for positioning Pc to a subset of its target genes.

Chromodomain protein CDYL is required for transmission/restoration of repressive histone marks

Faithful transmission or restoration of epigenetic information such as repressive histone modifications through generations is critical for the maintenance of cell identity. We report here that chromodomain Y-like protein (CDYL), a chromodomain-containing transcription corepressor, is physically associated with chromatin assembly factor 1 (CAF-1) and the replicative helicase MCM complex. We showed that CDYL bridges CAF-1 and MCM, facilitating histone transfer and deposition during DNA replication. We demonstrated that CDYL recruits histone-modifying enzymes G9a, SETDB1, and EZH2 to replication forks, leading to the addition of H3K9me2/3 and H3K27me2/3 on newly deposited histone H3. Significantly, depletion of CDYL impedes early S phase progression and sensitizes cells to DNA damage. Our data indicate that CDYL plays an important role in the transmission/restoration of repressive histone marks, thereby preserving the epigenetic landscape for the maintenance of cell identity.

CHD5 is a potential tumor suppressor in non small cell lung cancer (NSCLC)

Lung cancer is one of the deadliest types of cancers and genetic and epigenetic alterations play major roles in its development. Chromodomain (CHD) protein family acts in chromatin organization, regulation of transcription and also genomic stability and cancer prevention. Although CHD5, a member of this family was shown to contribute to major cellular events and functions as a tumor suppressor gene in various types of cancer, it is not clear whether CHD5 plays a role in lung carcinogenesis. The aim of this study was to investigate the possible role of CHD5 in progression of non-small cell lung cancer (NSCLC). Expression levels of CHD5 gene in 59 tumor and corresponding non-cancerous lung tissue samples were analyzed by qRT-PCR and the methylation status of the promoter region was investigated by methylation specific PCR (MS-PCR). The Akt phosphorylation levels were investigated by Western Blot (WB). CHD5 was down-regulated in 17 (39.5%) and up-regulated in 24 (55.8%) of tumor specimens. Even though the promoter of CHD5 was hypermethylated in 8 patients, it was not found associated with CHD5 gene expression (p=0.08). Akt phosphorylation was increased in 14 (53.8%) and decreased in 12 (46.2%) of the samples but no significant association was found between p-Akt phosphorylation and CHD5 expression (p=0.67). We suggest that CHD5 may act as a tumor suppressor gene in NSCLC.

A Novel Role of Chromodomain Protein CBX8 in DNA Damage Response

Induction of DNA damage induces a dynamic repair process involving DNA repair factors and epigenetic regulators. Chromatin alterations must occur for DNA repair factors to gain access to DNA lesions and restore original chromatin configuration to preserve the gene expression profile. We characterize the novel role of CBX8, a chromodomain-containing protein with established roles in epigenetic regulation in DNA damage response. CBX8 protein rapidly accumulates at the sites of DNA damage within 30 s and progresses to accumulate until 4 min before gradually dispersing back to its predamage distribution by 15 min. CBX8 recruitment to the sites of DNA damage is dependent upon PARP1 activation and not dependent on ATM activation. CBX8 biochemically interacts with TRIM33, and its recruitment to DNA damage is also dependent on the presence of TRIM33. Knockdown of CBX8 using siRNA significantly reduces the efficiency of both homologous and the other non-homologous recombination, as well as increases sensitivity of cells to ionizing radiation. These findings demonstrate that CBX8 functions in the PARP-dependent DNA damage response partly through interaction with TRIM33 and is required for efficient DNA repair.

Dual chromatin recognition by the histone deacetylase complex HCHC is required for proper DNA methylation in Neurospora crassa

DNA methylation, heterochromatin protein 1 (HP1), histone H3 lysine 9 (H3K9) methylation, histone deacetylation, and highly repeated sequences are prototypical heterochromatic features, but their interrelationships are not fully understood. Prior work showed that H3K9 methylation directs DNA methylation and histone deacetylation via HP1 in Neurospora crassa and that the histone deacetylase complex HCHC is required for proper DNA methylation. The complex consists of the chromodomain proteins HP1 and chromodomain protein 2 (CDP-2), the histone deacetylase HDA-1, and the AT-hook motif protein CDP-2/HDA-1-associated protein (CHAP). We show that the complex is required for proper chromosome segregation, dissect its function, and characterize interactions among its components. Our analyses revealed the existence of an HP1-based DNA methylation pathway independent of its chromodomain. The pathway partially depends on CHAP but not on the CDP-2 chromodomain. CDP-2 serves as a bridge between the recognition of H3K9 trimethylation (H3K9me3) by HP1 and the histone deacetylase activity of HDA-1. CHAP is also critical for HDA-1 localization to heterochromatin. Specifically, the CHAP zinc finger interacts directly with the HDA-1 argonaute-binding protein 2 (Arb2) domain, and the CHAP AT-hook motifs recognize heterochromatic regions by binding to AT-rich DNA. Our data shed light on the interrelationships among the prototypical heterochromatic features and support a model in which dual recognition by the HP1 chromodomain and the CHAP AT-hooks are required for proper heterochromatin formation.

EZH2 and BCL6 Cooperate to Assemble CBX8-BCOR Complex to Repress Bivalent Promoters, Mediate Germinal Center Formation and Lymphomagenesis

The EZH2 histone methyltransferase mediates the humoral immune response and drives lymphomagenesis through formation of bivalent chromatin domains at critical germinal center (GC) B cell promoters. Herein we show that the actions of EZH2 in driving GC formation and lymphoma precursor lesions require site-specific binding by the BCL6 transcriptional repressor and the presence of a non-canonical PRC1-BCOR-CBX8 complex. The chromodomain protein CBX8 is induced in GC B cells, binds to H3K27me3 at bivalent promoters, and is required for stable association of the complex and the resulting histone modifications. Moreover, oncogenic BCL6 and EZH2 cooperate to accelerate diffuse large B cell lymphoma (DLBCL) development and combinatorial targeting of these repressors results in enhanced anti-lymphoma activity in DLBCLs.

Expression level of chromodomain Y (CDY): potential marker for prediction of sperm recovery in non-obstructive azoospermia

The availability of testis specific genes will be of help in choosing the most promising biomarkers for the detection of testicular sperm retrieval in patients with non-obstructive azoospermia (NOA). Testis specific chromodomain protein Y 1 (CDY1) is a histone acetyltransferase which concentrates in the round spermatid nucleus, where histone hyperacetylation occurs and causes the replacement of histones by the sperm-specific DNA packaging proteins, TNPs and PRMs. The aim was to evaluate CDY1 gene as a marker for predicting of successful sperm retrieval in NOA patients. This research was conducted on 29 patients with NOA who had undergone testicular sperm extraction (TESE) procedure. NOA patients were subdivided into patients with successful sperm retrieval (NOA+, n=12) and patients with unsuccessful sperm retrieval (NOA-, n=17). Relative expression of CDY1 gene and chromatin incorporation of CDY1 protein were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and ELISA assay, respectively. Quantification of mRNA relative expression and incorporation of CDY1 protein in chromatin showed significant lower expressions and protein levels of CDY1 in testis tissues of NOA- in comparison to NOA+ group. The findings in this study demonstrated a correlation between the low levels of CDY1 function and unsuccessful sperm recovery in the testicular tissues of NOA- compared to NOA+ patients. Therefore, it can be reasonable to consider CDY1 as a potential biomarker for predicting the presence of spermatozoa, although the claim needs more samples to be confirmed.

Mechanism of chromatin segregation to the nuclear periphery in C. elegans embryos.

In eukaryotic organisms, gene regulation occurs in the context of chromatin. In the interphase nucleus, euchromatin and heterochromatin occupy distinct space during cell differentiation, with heterochromatin becoming enriched at the nuclear and nucleolar peripheries. This organization is thought to fine-tune gene expression. To elucidate the mechanisms that govern this level of genome organization, screens were carried out in C. elegans which monitored the loss of heterochromatin sequestration at the nuclear periphery. This led to the identification of a novel chromodomain protein, CEC-4 (Caenorhabditis elegans chromodomain protein 4) that mediates the anchoring of H3K9 methylation-bearing chromatin at the nuclear periphery in early to mid-stage embryos. Surprisingly, the loss of CEC-4 does not derepress genes found in heterochromatic domains, nor does it affect differentiation under standard laboratory conditions. On the other hand, CEC-4 contributes to the efficiency with which muscle differentiation is induced following ectopic expression of the master regulator, HLH-1. This is one of the first phenotypes specifically attributed to the ablation of heterochromatin anchoring.

CRL6, a member of the CHD protein family, is required for crown root development in rice

The root system in monocotyledonous plants is largely composed of postembryonic shoot-borne roots named crown roots, which are important for nutrients and water uptake. The molecular mechanism underlying regulation of crown root development is not fully explored. In this study, we characterized a rice (Oryza sativa) mutant defective in crown root formation, designated as crown rootless6 (crl6). Histological analysis showed that CRL6 influences crown root formation by regulating primordial initiation and development. Map-based cloning and subsequent complementation tests verified that the CRL6 gene encodes a member of the large chromodomain, helicase/ATPase, and DNA-binding domain (CHD) family protein. Realtime RT-PCR analysis showed that CRL6 was most highly expressed in the stem base region where crown roots initiated. In addition, auxin-action inhibited phenotype was observed during crl6 development. The expressions of OsIAA genes were down-regulated in crl6. Our results provide evidence that CRL6 plays an important role in crown root development in rice via auxin-related signaling pathway.

A Chromatin Reader That Acts As a Key to Lock in and Coordinate Recruitment of Transcription Factors and a Novel Polycomb Complex to Bivalent Chromatin Thus Driving Formation of Germinal Centers and B-Cell Lymphomas

The EZH2 Polycomb H3K27 histone methyltransferase and the BCL6 transcriptional repressor are highly expressed in germinal center (GC) B cells and are each targeted by activating somatic mutations in DLBCL. In previous work we demonstrated that i) EZH2 is required for GC formation, ii) EZH2 Y641 somatic mutation drives GC hyperplasia and lymphomagenesis and iii) that WT and mutant EZH2 function by creating bivalent chromatin at promoters of genes that regulate cell cycle and terminal differentiation. In other work we showed that BCL6 represses gene promoters specifically by recruiting BCOR complex (a non-canonical form of PRC1 complex); and that BCL6 and EZH2 bind to many of the same promoters, although they do not physically interact. Finally we reported that the presence of both EZH2 and BCL6 was required for BCOR complex to stably associate with chromatin. This led us to hypothesize that BCL6 and EZH2 may be mutually dependent in normal and malignant B-cells.We found that conditional deletion of EZH2 from already established GC B-cells abrogates the ability of constitutive IµBCL6 expression to induce GC hyperplasia. A similar effect was observed by administering the specific EZH2 inhibitor GSK503. For the reciprocal experiments we developed two new mouse models: a conditional BCL6 knockout and a conditional EZH2Y641F knockin in the EZH2 endogenous locus. We showed that EZH2Y641F knockin mice developed GC hyperplasia, but this effect is abrogated by conditional deletion of BCL6 from established GC B cells. Given cell line data suggesting that BCOR might be important for the effect of EZH2 and BCL6 we next generated GC BCOR conditional knockout mice and observed that loss of BCOR not only disrupts GC formation but also abrogates the GC hyperplasia when crossed into the conditional EZH2Y641F knockin mice. Reversal of the EZH2Y641F knockin GC hyperplasia was also achieved by treating these mice with a novel small molecule called 1085 that potently disrupts BCL6 binding to BCOR. Notably, 1085 synergized with EZH2 inhibitors to kill DLBCL cells in vitro and eradicate DLBCL xenografts in vivo. Collectively these data show that EZH2, BCL6 and BCOR are mutually dependent both in the context of the normal GC reaction as well as in the setting of lymphoma precursor lesions driven by constitutive BCL6 expression or mutant EZH2.We next wished to determine the basic mechanism that explains the functional connection between EZH2 and BCL6-BCOR. Using ChIP-seq in primary human GC B cells we showed that there is significant overlap between BCL6-BCOR complex binding with EZH2 and bivalent chromatin. Using ChIP-re-ChIP we found that EZH2, BCOR and BCL6 are all localized to the same chromatin at bivalent gene promoters. Previously we showed that EZH2 mediated H3K27me3 is required for BCOR recruitment. However it was not clear why or how H3K27me3 is important for this effect. We postulated that a histone reader protein might bind to BCOR and recruit it to H3K27me3 marked chromatin. We therefore performed tandem affinity purification of BCOR followed by mass spectrometry to identify putative partner proteins and identified the chromodomain containing protein CBX8 as a novel BCOR partner. Notably CBX8 is the only H3K27 chromodomain protein that is upregulated in GC B cells. We confirmed endogenous BCOR-CBX8 interaction in human DLBCL cells. We showed that CBX8 is recruited to bivalent chromatin in an H3K27me3 dependent manner, and CBX8 depletion results in loss of BCOR complex assemble. Most strikingly, CBX8 knockdown was a phenocopy of EZH2 shRNA or EZH2 inhibitor. CBX8 loss resulted in derepression of EZH2 targets, and phenocopied the effect of EZH2 shRNA or inhibitors (proliferation arrest followed by plasma cell differentiation and then apoptosis). Therefore, CBX8 is shown for the first time to recruit non-canonical PRC1 and to explain how EZH2 cooperates with BCL6 to repress transcription and mediate the GC and DLBCL phenotype. PRC2 functions in a mutually interdependent manner with BCL6 to jointly enable stable association of non-canonical PRC1 complex with bivalent chromatin.In summary, our data establish a paradigm whereby EZH2 functions in a coordinate manner with transcription factors to transiently suppress genes in a cell context-specific manner relevant to its function in normal immunity as well as DLBCL. Our results also provide a rational basis for clinical translation of combined EZH2 and BCL6 inhibitors. DisclosuresLevine:Foundation Medicine: Consultancy; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees.

Perinuclear Anchoring of H3K9-Methylated Chromatin Stabilizes Induced Cell Fate in C. elegans Embryos

Interphase chromatin is organized in distinct nuclear sub-compartments, reflecting its degree of compaction and transcriptional status. In Caenorhabditis elegans embryos, H3K9 methylation is necessary to silence and to anchor repeat-rich heterochromatin at the nuclear periphery. In a screen for perinuclear anchors of heterochromatin, we identified a previously uncharacterized C.elegans chromodomain protein, CEC-4. CEC-4 binds preferentially mono-, di-, or tri-methylated H3K9 and localizes at the nuclear envelope independently of H3K9 methylation and nuclear lamin. CEC-4 is necessary for endogenous heterochromatin anchoring, but not for transcriptional repression, in contrast to other known H3K9 methyl-binders in worms, which mediate gene repression but not perinuclear anchoring. When we ectopically induce a muscle differentiation program in embryos, cec-4 mutants fail to commit fully to muscle cell fate. This suggests that perinuclear sequestration of chromatin during development helps restrict cell differentiation programs by stabilizing commitment to a specific cell fate. PAPERCLIP.

A Phosphotyrosine Switch Controls the Association of Histone Mark Readers with Methylated Proteins.

Although histone post-translational modifications play a paramount role in controlling access to genetic information, our understanding of the precise mechanisms regulating chromatin signaling remains superficial. For instance, histone H3 trimethylated on lysine 9 (H3K9(me3)) favors the association of chromodomain proteins such as heterochromatin protein 1α (HP1α) with chromatin. However, HP1α and other such chromatin proteins are not covering all specific histone marks at all times. Thus, how are these reader-histone interactions regulated? We propose tyrosine phosphorylation within the aromatic cage of histone mark readers as a molecular switch that can either turn ON or OFF and even alter the specificity of reader-histone interactions. We have identified tyrosine phosphorylation events on the chromatin proteins HP1α and M-phase phosphoprotein 8 that regulate their association with methylated histones in vitro (synthetic peptides, calf thymus purified histones, and nucleosomes), but also in cells, thus controlling access to genetic information.

Restricted distribution of mrg-1 mRNA in C. elegans primordial germ cells through germ granule-independent regulation.

The chromodomain protein MRG-1 is an essential maternal factor for proper germline development that protects germ cells from cell death in C. elegans. Unlike germ granules, which are exclusively segregated to the germline blastomeres at each cell division from the first cleavage of the embryo, MRG-1 is abundant in all cells in early embryos and is then gradually restricted to the primordial germ cells (PGCs) by the morphogenesis stage. Here, we show that this characteristic spatiotemporal expression pattern is dictated by the mrg-1 3'UTR and is differentially regulated at the RNA level between germline and somatic cells. Asymmetric segregation of germ granules is not necessary to localize MRG-1 to the PGCs. We found that MES-4, an essential chromatin regulator in germ cells, also accumulates in the PGCs in a germ granule-independent manner. We propose that C.elegans PGCs have a novel mechanism to accumulate at least some chromatin-associated proteins that are essential for germline immortality.

Chromodomain protein Tcd1 is required for macronuclear genome rearrangement and repair in Tetrahymena.

The survival of an organism’s progeny depends on the maintenance of its genome. Programmed DNA rearrangement and repair in Tetrahymena occur during the differentiation of the developing somatic macronuclear genome from the germ line micronuclear genome. Tetrahymena chromodomain protein (Tcd1) exhibited dynamic localization from the parental to the developing macronuclei. In the developing macronuclei, Tcd1 colocalized with Pdd1 and H3K9me3. Furthermore, Tcd1 colocalized with Pdd1 in the conjusome and “donut structure” of DNA elimination heterochromatin region. During the growth and conjugation stages, TCD1 knockout cells appeared normal and similar to wild-type strains. In addition, these knockout cells proceeded to the 2MAC-1MIC stage. However, the progeny of the TCD1 knockout cells did not grow upon return to SPP medium and eventually died. The deletion of the internal elimination sequence R element was partially disrupted in the developing new macronuclei. Gamma H2A staining showed that Tcd1 loss induced the accumulation of DNA double-strand breaks and the failure of genome repair. These results suggest that the chromodomain protein Tcd1 is required for the rearrangement and repair of new macronuclear genome in Tetrahymena.

Proteasome regulation of the chromodomain protein MRG-1 controls the balance between proliferative fate and differentiation in the C. elegans germ line.

The level of stem cell proliferation must be tightly controlled for proper development and tissue homeostasis. Multiple levels of gene regulation are often employed to regulate stem cell proliferation to ensure that the amount of proliferation is aligned with the needs of the tissue. Here we focus on proteasome-mediated protein degradation as a means of regulating the activities of proteins involved in controlling the stem cell proliferative fate in the C. elegans germ line. We identify five potential E3 ubiquitin ligases, including the RFP-1 RING finger protein, as being involved in regulating proliferative fate. RFP-1 binds to MRG-1, a homologue of the mammalian chromodomain-containing protein MRG15 (MORF4L1), which has been implicated in promoting the proliferation of neural precursor cells. We find that C. elegans with reduced proteasome activity, or that lack RFP-1 expression, have increased levels of MRG-1 and a shift towards increased proliferation in sensitized genetic backgrounds. Likewise, reduction of MRG-1 partially suppresses stem cell overproliferation. MRG-1 levels are controlled independently of the spatially regulated GLP-1/Notch signalling pathway, which is the primary signal controlling the extent of stem cell proliferation in the C. elegans germ line. We propose a model in which MRG-1 levels are controlled, at least in part, by the proteasome, and that the levels of MRG-1 set a threshold upon which other spatially regulated factors act in order to control the balance between the proliferative fate and differentiation in the C. elegans germ line.

The spindle matrix protein, Chromator, is a novel tubulin binding protein that can interact with both microtubules and free tubulin.

The chromodomain protein, Chromator, is localized to chromosomes during interphase; however, during cell division together with other nuclear proteins Chromator redistributes to form a macro molecular spindle matrix complex that embeds the microtubule spindle apparatus. It has been demonstrated that the CTD of Chromator is sufficient for localization to the spindle matrix and that expression of this domain alone could partially rescue Chro mutant microtubule spindle defects. Furthermore, the presence of frayed and unstable microtubule spindles during mitosis after Chromator RNAi depletion in S2 cells indicated that Chromator may interact with microtubules. In this study using a variety of biochemical assays we have tested this hypothesis and show that Chromator not only has binding activity to microtubules with a Kd of 0.23 µM but also to free tubulin. Furthermore, we have mapped the interaction with microtubules to a relatively small stretch of 139 amino acids in the carboxy-terminal region of Chromator. This sequence is likely to contain a novel microtubule binding interface since database searches did not find any sequence matches with known microtubule binding motifs.

Deletion of CDY1b copy of Y chromosome CDY1 gene is a risk factor of male infertility in Tunisian men

The relationship between male infertility and microdeletions in the Y chromosome that remove multiple genes varies among countries and populations. The aim of this study was to investigate the different types of Chromodomain protein, Y-linked 1 (CDY1) gene deletions and their effect on male infertility and spermatogenesis in Tunisian men. A total of 241 infertile men with different spermatogenic impairments and 115 fertile men were included in this study. We determined the prevalence of CDY1a and CDY1b copy deletions by PCR-RFLP using PvuII as restriction endonuclease. Results: Among the 356 Tunisian individuals, 93.25% had the two copies (CDY1a and CDY1b) of CDY gene (91.2% in infertile patients and 97.3% in fertile men). We also found that deletion of CDY1b was significantly more frequent in infertile patients (azoo/oligospermic and normospermic) than in fertile men (7% vs 1.7% respectively; p value=0.02). However, deletion of CDY1a copy was very rare, and was detected in only one fertile man and four normospermic infertile patients. Our findings showed that deletion of CDY1b copy gene is a significant risk factor for male infertility independent of sperm concentration, whereas deletion of CDY1a gene seems to have no effect on fertility in the Tunisian population.

Eaf5/7/3 form a functionally independent NuA4 submodule linked to RNA polymerase II-coupled nucleosome recycling.

The NuA4 histone acetyltransferase complex is required for gene regulation, cell cycle progression, and DNA repair. Dissection of the 13-subunit complex reveals that the Eaf7 subunit bridges Eaf5 with Eaf3, a H3K36me3-binding chromodomain protein, and this Eaf5/7/3 trimer is anchored to NuA4 through Eaf5. This trimeric subcomplex represents a functional module, and a large portion exists in a native form outside the NuA4 complex. Gene-specific and genome-wide location analyses indicate that Eaf5/7/3 correlates with transcription activity and is enriched over the coding region. In agreement with a role in transcription elongation, the Eaf5/7/3 trimer interacts with phosphorylated RNA polymerase II and helps its progression. Loss of Eaf5/7/3 partially suppresses intragenic cryptic transcription arising in set2 mutants, supporting a role in nucleosome destabilization. On the other hand, loss of the trimer leads to an increase of replication-independent histone exchange over the coding region of transcribed genes. Taken together, these results lead to a model where Eaf5/7/3 associates with elongating polymerase to promote the disruption of nucleosomes in its path, but also their refolding in its wake.

Coordinated Regulation of Dendrite Arborization by Epigenetic Factors CDYL and EZH2

Dendritic arborization is one of the key determinants of precise circuits for information processing in neurons. Unraveling the molecular mechanisms underlying dendrite morphogenesis is critical to understanding the establishment of neuronal connections. Here, using gain- and loss-of-function approaches, we defined the chromodomain protein and transcription corepressor chromodomain Y-like (CDYL) protein as a negative regulator of dendrite morphogenesis in rat/mouse hippocampal neurons both in vitro and in vivo. Overexpressing CDYL decreased, whereas knocking it down increased, the dendritic complexity of the primary cultured rat neurons. High-throughput DNA microarray screening identified a number of CDYL downstream target genes, including the brain-derived neurotrophic factor (BDNF). Knock-down of CDYL in neuronal cells led to increased expression of BDNF, which is primarily responsible for CDYL's effects on dendrite patterns. Mechanistically, CDYL interacts with EZH2, the catalytic subunit of Polycomb Repressive Complex 2 (PRC2), directly and recruits the H3K27 methyltransferase activity to the promoter region of the BDNF gene. In doing so, CDYL and EZH2 coordinately restrict dendrite morphogenesis in an interdependent manner. Finally, we found that neural activity increased dendritic complexity through degradation of CDYL protein to unleash its inhibition on BDNF. These results link, for the first time, the epigenetic regulators CDYL and EZH2 to dendrite morphogenesis and might shed new light on our understanding of the regulation of the neurodevelopment.