The novel BRDT inhibitor NHWD870 shows potential as a male contraceptive in mice.

Small molecule inhibitors of the bromodomain and extraterminal domain (BET) family proteins have emerged as promising options not only for the treatment of multiple cancers but also for disturbing the process of sperm maturation with potential for use as viable contraceptive targets. In this study, we find that the BET family inhibitor NHWD870 and BRDT can bind well in vitro through bioinformatics software prediction and protein binding inhibition experiments. NHWD870 can produce a good contraceptive effect through animal experiments in vivo, and the fertility can be restored to normal after drug withdrawal. Transcriptomics and proteomics results suggest that NHWD870 affects pathways related to spermatogenesis and maturation, further contributing to the male infertility phenotype. Our results show that NHWD870 can induce a complete and reversible contraceptive effect in mice, which is stronger than that of JQ1 and its synthesized derivatives. This study is expected to eventually lead to clinical trials.

Discovery of potent BET bromodomain 1 stereoselective inhibitors using DNA-encoded chemical library selections.

BRDT, BRD2, BRD3, and BRD4 comprise the bromodomain and extraterminal (BET) subfamily which contain two similar tandem bromodomains (BD1 and BD2). Selective BD1 inhibition phenocopies effects of tandem BET BD inhibition both in cancer models and, as we and others have reported of BRDT, in the testes. To find novel BET BD1 binders, we screened >4.5 billion molecules from our DNA-encoded chemical libraries with BRDT-BD1 or BRDT-BD2 proteins in parallel. A compound series enriched only by BRDT-BD1 was resynthesized off-DNA, uncovering a potent chiral compound, CDD-724, with >2,000-fold selectivity for inhibiting BRDT-BD1 over BRDT-BD2. CDD-724 stereoisomers exhibited remarkable differences in inhibiting BRDT-BD1, with the R-enantiomer (CDD-787) being 50-fold more potent than the S-enantiomer (CDD-786). From structure­activity relationship studies, we produced CDD-956, which maintained picomolar BET BD1 binding potency and high selectivity over BET BD2 proteins and had improved stability in human liver microsomes over CDD-787. BROMOscan profiling confirmed the excellent pan-BET BD1 affinity and selectivity of CDD-787 and CDD-956 on BD1 versus BD2 and all other BD-containing proteins. A cocrystal structure of BRDT-BD1 bound with CDD-956 was determined at 1.82 Å and revealed BRDT-BD1­specific contacts with the αZ and αC helices that explain the high affinity and selectivity for BET BD1 versus BD2. CDD-787 and CDD-956 maintain cellular BD1-selectivity in NanoBRET assays and show potent antileukemic activity in acute myeloid leukemia cell lines. These BET BD1-specific and highly potent compounds are structurally unique and provide insight into the importance of chirality to achieve BET specificity.

NUT Is a Driver of p300-Mediated Histone Hyperacetylation: From Spermatogenesis to Cancer.

In maturing sperm cells, a major genome re-organization takes place, which includes a global increase in the acetylation of histones prior to their replacement by protamines, the latter being responsible for the tight packaging of the male genome. Understanding the function of the oncogenic BRD4-NUT fusion protein in NUT carcinoma (NC) cells has proven to be essential in uncovering the mechanisms underlying histone hyperacetylation in spermatogenic cells. Indeed, these studies have revealed the mechanism by which a cooperation between BRD4, a bromodomain factor of the BET family, NUT, a normally testis-specific factor, and the histone acetyltransferase p300, induces the generation of hyperacetylated chromatin domains which are present in NC cells. The generation of Nut ko mice enabled us to demonstrate a genetic interaction between Nut and Brdt, encoding BRDT, a testis-specific BRD4-like factor. Indeed, in spermatogenic cells, NUT and p300 interact, which results in an increased acetylation of histone H4 at both positions K5 and K8. These two positions, when both acetylated, are specifically recognized by the first bromodomain of BRDT, which then mediates the removal of histone and their replacement by protamines. Taken together, these investigations show that the fusion of NUT to BRD4 in NUT Carcinoma cells reconstitutes, in somatic cells, a functional loop, which normally drives histone hyperacetylation and chromatin binding by a BET factor in spermatogenic cells.

Epigenetic Dysregulation of BRDT Gene in Testis Tissues of Infertile Men: Case-Control Study.

Objective: Bromodomain testis associated (BRDT), a testis-specific member of the Bromo- and Extra-Terrminal domain (BET) protein family, is involved in spermatogenesis and, more specifically, chromatin remodeling. In the post-meiotic spermatogenic cells, BRDT protein binds to the hyperacetylated histones and facilitates their replacement with transition proteins (TPs), particularly protamines, which are essential for chromatin condensation. The current research was conducted to assess the expression and epigenetic profile of BRDT in the testis tissues of infertile men. Materials and Methods: In this case-control study, three groups were included: positive control group: obstructive azoospermia (OA, n=10), round spermatid maturation arrest group (SMA, n=10) and negative control group: sertoli cellonly syndrome (SCOS, n=10). Using quantitative real-time polymerase chain reaction (PCR), the expression profile of BRDT was generated. Also, ChIP-real time PCR was used to measure the following histone marks: H3K9ac, H3K9me3, H3K4me3, H3K27me3 on the promoter region of BRDT. Results: Our data indicated that BRDT expression decreased in the SMA group in comparison with the positive control group and this finding is in line with the ChIP results obtained in this group. Conclusion: Based on these data, we postulate that BRDT gene has a vital role in the spermatogenesis and its decreased expression due to an aberrant epigenetic signaling might be associated with male infertility.

Relation of gilthead seabream (Sparus aurata) seasonal reproductive activity to hematology, serum biochemistry, histopathology, and Brdt gene expression.

This study aimed to find the relation of Sparus aurata (gilthead seabream) reproductive activities to some blood parameters as complete blood count, liver enzymes, some hormones related to reproduction process and microscopic findings of gonads, as well as expression of Bromodomain testis-specific gene. Eighty-eight sexually mature seabream were collected and investigated through the four seasons. Red blood cells were higher in autumn and spring. Hemoglobin was high in summer, MCV highest values ​​were seen in winter and summer, while MCHC was highest in summer. The values ​​of white blood cells increased significantly in spring, summer, and autumn compared with winter. The highest value of lymphocytes was recorded in spring and autumn. Eosinophil was recorded the highest value in the spring. The highest value of segmented neutrophils was recorded in summer. The highest value of band neutrophil was recorded in summer and winter. Alanine aminotransferase and aspartate aminotransferase showed high values in the winter. Luteinizing hormone (LH) was higher in females, males, and hermaphrodites during winter. Follicle-stimulating hormone (FSH) was higher in females during spring. The highest value of estradiol 17-ß and progesterone was recorded in summer. The highest value of total testosterone was recorded in spring. Microscopically, ovaries were immature and inactive during spring and summer but well developed in autumn and winter. During spring and summer, testes were immature and began spermatogenesis process but well developed with the appearance of spermatids and spermatozoa during autumn and winter. The expression of Brdt was higher in testes than ovary. Brdt recorded high expression in autumn and spring than in summer and winter.

Genome-wide chromatin occupancy of BRDT and gene expression analysis suggest transcriptional partners and specific epigenetic landscapes that regulate gene expression during spermatogenesis.

BRDT, a member of the BET family of double bromodomain-containing proteins, is essential for spermatogenesis in the mouse and has been postulated to be a key regulator of transcription in meiotic and post-meiotic cells. To understand the function of BRDT in these processes, we first characterized the genome-wide distribution of the BRDT binding sites, in particular within gene units, by ChIP-Seq analysis of enriched fractions of pachytene spermatocytes and round spermatids. In both cell types, BRDT binding sites were mainly located in promoters, first exons, and introns of genes. BRDT binding sites in promoters overlapped with several histone modifications and histone variants associated with active transcription, and were enriched for consensus sequences for specific transcription factors, including MYB, RFX, ETS, and ELF1 in pachytene spermatocytes, and JunD, c-Jun, CRE, and RFX in round spermatids. Subsequent integration of the ChIP-seq data with available transcriptome data revealed that stage-specific gene expression programs are associated with BRDT binding to their gene promoters, with most of the BDRT-bound genes being upregulated. Gene Ontology analysis further identified unique sets of genes enriched in diverse biological processes essential for meiosis and spermiogenesis between the two cell types, suggesting distinct developmentally stage-specific functions for BRDT. Taken together, our data suggest that BRDT cooperates with different transcription factors at distinctive chromatin regions within gene units to regulate diverse downstream target genes that function in male meiosis and spermiogenesis.

PHF7 Modulates BRDT Stability and Histone-to-Protamine Exchange during Spermiogenesis.

Spermatogenesis is a complex process of sperm generation, including mitosis, meiosis, and spermiogenesis. During spermiogenesis, histones in post-meiotic spermatids are removed from chromatin and replaced by protamines. Although histone-to-protamine exchange is important for sperm nuclear condensation, the underlying regulatory mechanism is still poorly understood. Here, we identify PHD finger protein 7 (PHF7) as an E3 ubiquitin ligase for histone H3K14 in post-meiotic spermatids. Generation of Phf7-deficient mice and Phf7 C160A knockin mice with impaired E3 ubiquitin ligase activity reveals defects in histone-to-protamine exchange caused by dysregulation of histone removal factor Bromodomain, testis-specific (BRDT) in early condensing spermatids. Surprisingly, E3 ubiquitin ligase activity of PHF7 on histone ubiquitination leads to stabilization of BRDT by attenuating ubiquitination of BRDT. Collectively, our findings identify PHF7 as a critical factor for sperm chromatin condensation and contribute to mechanistic understanding of fundamental phenomenon of histone-to-protamine exchange and potential for drug development for the male reproduction system.

The Cannabinoid Receptor CB1 Stabilizes Sperm Chromatin Condensation Status During Epididymal Transit by Promoting Disulphide Bond Formation.

The cannabinoid receptor CB1 regulates differentiation of spermatids. We recently characterized spermatozoa from caput epididymis of CB1-knock-out mice and identified a considerable number of sperm cells with chromatin abnormality such as elevated histone content and poorly condensed chromatin. In this paper, we extended our findings and studied the role of CB1 in the epididymal phase of chromatin condensation of spermatozoa by analysis of spermatozoa from caput and cauda epididymis of wild-type and CB1-knock-out mouse in both a homozygous or heterozygous condition. Furthermore, we studied the impact of CB1-gene deletion on histone displacement mechanism by taking into account the hyperacetylation of histone H4 and players of displacement such as Chromodomain Y Like protein (CDYL) and Bromodomain testis-specific protein (BRDT). Our results show that CB1, via local and/or endocrine cell-to-cell signaling, modulates chromatin remodeling mechanisms that orchestrate a nuclear condensation extent of mature spermatozoa. We show that CB1-gene deletion affects the epididymal phase of chromatin condensation by interfering with inter-/intra-protamine disulphide bridges formation, and deranges the efficiency of histone removal by reducing the hyper-acetylation of histone H4. This effect is independent by gene expression of Cdyl and Brdt mRNA. Our results reveal a novel and important role for CB1 in sperm chromatin condensation mechanisms.

Metabolism of JQ1, an inhibitor of bromodomain and extra terminal bromodomain proteins, in human and mouse liver microsomes†.

JQ1 is a small-molecule inhibitor of the bromodomain and extra terminal (BET) protein family that potently inhibits the bromodomain testis-specific protein (BRDT), which is essential for spermatogenesis. JQ1 treatment produces a reversible contraceptive effect by targeting the activity of BRDT in mouse male germ cells, validating BRDT as a male contraceptive target. Although JQ1 possesses favourable physical properties, it exhibits a short half-life. Because the details of xenobiotic metabolism play important roles in the optimization of drug candidates and in determining the role of metabolism in drug efficacy, we investigated the metabolism of JQ1 in human and mouse liver microsomes. We present the first comprehensive view of JQ1 metabolism in liver microsomes, distinguishing nine JQ1 metabolites, including three monohydroxylated, one de-tert-butylated, two dihydroxylated, one monohydroxylated/dehydrogenated, one monohydroxylated-de-tert-butylated and one dihydroxylated/dehydrogenated variant of JQ1. The dominant metabolite (M1) in both human and mouse liver microsomes is monohydroxylated on the fused three-ring core. Using recombinant cytochrome P450 (CYP) enzymes, chemical inhibitors and the liver S9 fraction of Cyp3a-null mice, we identify enzymes that contribute to the formation of these metabolites. Cytochrome P450 family 3 subfamily A member 4 (CYP3A4) is the main contributor to the production of JQ1 metabolites in vitro, and the CYP3A4/5 inhibitor ketoconazole strongly inhibits JQ1 metabolism in both human and mouse liver microsomes. Our findings suggest that JQ1 half-life and efficacy might be improved in vivo by co-administration of a selective CYP inhibitor, thereby impacting the use of JQ1 as a probe for BRDT activity in spermatogenesis and as a probe or therapeutic in other systems.

[Advances in the molecular genetic studies of acephalic spermatozoa syndrome].

Acephalic spermatozoa syndrome (ASS) is characterized by a predominance of headless spermatozoa with abnormal head-tail junction in the ejaculate, which causes severe male infertility. The pathogenic mechanism of ASS remained unclarified for a long time until recent identification of the four ASS-associated genes SUN5, PMFBP1, TSGA10, and BRDT and their mutations due to the development of high-throughput sequencing technology. This review summarizes the advances in the genetic studies of ASS, focusing on its pathogenic molecular mechanisms, which provide an important basis for the molecular diagnosis of the disease as well as for assisted reproductive technology.

Histone Variant H2A.L.2 Guides Transition Protein-Dependent Protamine Assembly in Male Germ Cells.

Histone replacement by transition proteins (TPs) and protamines (Prms) constitutes an essential step for the successful production of functional male gametes, yet nothing is known on the underlying functional interplay between histones, TPs, and Prms. Here, by studying spermatogenesis in the absence of a spermatid-specific histone variant, H2A.L.2, we discover a fundamental mechanism involved in the transformation of nucleosomes into nucleoprotamines. H2A.L.2 is synthesized at the same time as TPs and enables their loading onto the nucleosomes. TPs do not displace histones but rather drive the recruitment and processing of Prms, which are themselves responsible for histone eviction. Altogether, the incorporation of H2A.L.2 initiates and orchestrates a series of successive transitional states that ultimately shift to the fully compacted genome of the mature spermatozoa. Hence, the current view of histone-to-nucleoprotamine transition should be revisited and include an additional step with H2A.L.2 assembly prior to the action of TPs and Prms.

Whole-exome sequencing identified a homozygous BRDT mutation in a patient with acephalic spermatozoa.

Acephalic spermatozoa is a very rare disorder of male infertility. Here, in a patient from from a consanguineous family, we have identified, by whole-exome sequencing, a homozygous mutation (c.G2783A, p.G928D) in the BRDT gene. The gene product, BRDT, is a testis-specific protein that is considered an important drug target for male contraception. The G928D mutation is in the P-TEFb binding domain, which mediates the interaction with transcription elongation factor and might affect the transcriptional activities of downstream genes. By RNA-sequencing analysis of cells expressing the BRDT mutation, we found the p.G928D mutation protein causes mis-regulation of 899 genes compared with BRDT wild-type cells. Furthermore, by Gene Ontology analysis, the upregulated genes in p.G928D cells were enriched in the processes of intracellular transport, RNA splicing, cell cycle and DNA metabolic process, revealing the underlying mechanism of the pathology that leads to acephalic spermatozoa.

[Male contraception - the current state of knowledge]. / Antykoncepcja meska - aktualny stan wiedzy.

Contraception is important from a health, psychological and socioeconomic point of view. Due to the fact that male-based contraceptive methods are mostly represented by condoms and vasectomy, researchers are working on the new solutions, which could let the men be more involved in a conscious family planning. In this review we will present the current state of knowledge on this subject. There is a lot going on in the field of hormonal contraception. Studies including testosterone, progestins, synthetic androgens and other derivatives are on a different stages of clinical trials and mostly demonstrate high efficacy rates. Recent discovers of Izumo and Juno proteins, essential for the fertilization process, give hope for an easily reversible, non-hormonal method. Researchers are also trying to interfere with the process of spermatogenesis using BRDT inhibitor - JQ1, or neutralize the sperm by injecting styrene maleic anhydride (SMA) into the lumen of the vas deferens. The other studies explore processes involved in proper sperm motility. A vaccine which induces an immune response to the reproductive system is also an interesting method. The latest research use ultrasound waves and mechanical device which blocks the patency of vas deferens. The aim of the study current state of knowledge male contraception.

Small-Molecule Targeting of BET Proteins in Cancer.

BET proteins have recently become recognized for their role in a broad range of cancers and are defined by the presence of two acetyl-histone reading bromodomains and an ET domain. This family of proteins includes BRD2, BRD3, BRD4, and BRDT. BRD4 is the most-studied BET protein in cancer, and normally serves as an epigenetic reader that links active chromatin marks to transcriptional elongation through activation of RNA polymerase II. The role of BRD3 and BRD4 first became known in cancer as mutant oncoproteins fused to the p300-recruiting NUT protein in a rare aggressive subtype of squamous cell cancer known as NUT midline carcinoma (NMC). BET inhibitors are acetyl-histone mimetics that specifically bind BET bromodomains, competitively inhibiting its engagement with chromatin. The antineoplastic effects of BET inhibitors were first demonstrated in NMC and have since been shown to be effective at inhibiting the growth of many different cancers, particularly acute leukemia. BET inhibitors have also been instrumental as tool compounds that have demonstrated the key role of BRD4 in driving NMC and non-NMC cancer growth. Many clinical trials enrolling patients with hematologic and solid tumors are ongoing, with encouraging preliminary findings. BET proteins BRD2, BRD3, and BRD4 are expressed in nearly all cells of the body, so there are concerns of toxicity with BET inhibitors, as well as the development of resistance. Toxicity and resistance may be overcome by combining BET inhibitors with other targeted inhibitors, or through the use of novel BET inhibitor derivatives.

New insights into the role of the Brdt protein in the regulation of development and spermatogenesis in the mouse.

The bromodomain testis-specific (BRDT) protein belongs to the bromodomain extra-terminal (BET) family of proteins. It serves as a transcriptional regulator of gene expression during spermatogenesis, and is an essential factor for the normal spermatogenesis process. In this study, we characterized mice of several age groups who lacked the Brdt gene. The testes of Brdt mutant mice aged 8 weeks exhibited complete spermatocyte maturation arrest with a significantly increased number of apoptotic cells. The weights of the testes and accessory glands as well as the testosterone levels of the mutant mice were significantly lower compared to the normal mice. The mutant mice had delayed puberty, with normal levels of testosterone and accessory gland weights at the age of 14 and 28 weeks. The testes of the mutant mice at older ages also exhibited round spermatids. The presence of the BRDT protein was identified in the mice pituitary gland. Microarray analysis of mice pituitaries showed that 28 genes were down-regulated while 26 genes were up-regulated in the absence of the Brdt gene. Our results suggest that in addition to its critical role in the spermatogenesis process, the BRDT protein is also responsible for scheduling male puberty by regulation of the pituitary-gonad axis.

BET Protein BRDT Complexes With HDAC1, PRMT5, and TRIM28 and Functions in Transcriptional Repression During Spermatogenesis.

The expression of BRDT, a member of the BET sub-family of double bromodomain-containing proteins, is restricted to the male germ line, specifically to pachytene-diplotene spermatocytes and early spermatids. We previously showed that loss of the first bromodomain of BRDT by targeted mutagenesis (Brdt(ΔBD1) ) resulted in sterility and abnormalities in spermiogenesis, but little is known about BRDT's function at the molecular level. As part of studies designed to identify BRDT-interacting proteins we stably introduced a FLAG-tagged BRDT cDNA into 293T cells, which do not normally express BRDT. Affinity-purification of FLAG-tagged BRDT complexes indicated that BRDT has novel interactions with the histone deacetylase HDAC1, the arginine-specific histone methyltransferase 5 PRMT5, and the Tripartite motif-containing 28 protein TRIM28. Immunofluorescent microscopy revealed that BRDT co-localized with each of these proteins in round spermatids and co-immunoprecipitation of testicular extracts showed that these proteins interact with BRDT. Furthermore, they bind the promoter of H1t, a putative target of BRDT-containing complexes. This binding of H1t was lost in mice expressing the Brdt(ΔBD1) mutant protein and concomitantly, H1t expression was elevated in round spermatids. Our study reveals a role for BRDT-containing complexes in the repression of gene expression in vivo that correlates with dramatic effects on chromatin remodeling and the progression of spermiogenesis.

Inhibition of BET bromodomains as a therapeutic strategy for cancer drug discovery.

As a conserved protein interaction module that recognizes and binds to acetylated lysine, bromodomain (BRD) contains a deep, largely hydrophobic acetyl lysine binding site. Proteins that share the feature of containing two BRDs and an extra-terminal domain belong to BET family, including BRD2, BRD3, BRD4 and BRDT. BET family proteins perform transcription regulatory function under normal conditions, while in cancer, they regulate transcription of several oncogenes, such as c-Myc and Bcl-2. Thus, targeting BET proteins may be a promising strategy, and intense interest of BET proteins has fueled the development of structure-based bromodomain inhibitors in cancer. In this review, we focus on summarizing several small-molecule BET inhibitors and their relevant anti-tumor mechanisms, which would provide a clue for exploiting new targeted BET inhibitors in the future cancer therapy.

Genetic association study of RNF8 and BRDT variants with non-obstructive azoospermia in the Chinese Han population.

Increasing evidence indicates that polymorphisms in genes relevant to spermatogenesis might modulate the efficiency of reproduction in men. Ring finger protein 8 (RNF8) and bromodomain testis-specific (BRDT) are two candidate genes associated with spermatogenesis. Here, we considered potential associations of 14 single nucleotide polymorphisms (SNPs) in RNF8 and BRDT genes in Chinese patients with non-obstructive azoospermia (NOA). We analyzed 361 men with NOA and 368 fertile controls by using Sequenom iplex technology. Our data did not reveal any variants associated with NOA susceptibility. However, we observed that rs104669 and rs195432 of RNF8 were in strong linkage disequilibrium. Haplotype analysis of the two SNPs indicated that the haplotype AC reduced the risk of NOA and the haplotype TC significantly evaluated the risk of NOA. Moreover, the RNF8 variants rs195432 (C/A p = 0.030), rs195434 (T/C p = 0.025), and rs2284922 (T/C p = 0.034) were correlated with the smaller testis volume.

BRDT gene sequence in human testicular pathologies and the implication of its single nucleotide polymorphism (rs3088232) on fertility.

Bromodomain testis-specific (BRDT) protein is essential for the normal process of spermatogenesis. Mutant mice that expressed truncated BRDT had impaired testicular histology with severely reduced sperm concentration and abnormal sperm morphology, while a model of knockout Brdt mice with no BRDT protein had complete meiotic arrest. A BRDT single nucleotide polymorphism (SNP) (rs3088232) was reported as being associated with infertility in men. We assessed testicular specimens of 276 azoospermic men who underwent testicular sperm extraction to search for specimens that showed spermatogenic impairments similar to those of mutant BRDT mice. Ten similar specimens were selected for BRDT gene sequencing and they revealed three NCBI-reported SNPs (rs10783071, rs3088232 and rs10747493) variously distributed among them. Bioinformatics analysis predicted that they would not affect protein activity. Further assessment of rs3088232 frequency in a large group of non-obstructive azoospermia men and fertile controls demonstrated no significant difference between them (27.2 and 21.7% respectively; p = 0.122, Fisher's exact test). We conclude that the testicular impairments observed in the 10 specimens were not a consequence of BRDT gene mutation. The association between BRDT rs3088232 and infertility that had been reported in other studies was not supported.

Genome-scale acetylation-dependent histone eviction during spermatogenesis.

A genome-wide histone hyperacetylation is known to occur in the absence of transcription in haploid male germ cells, spermatids, before and during the global histone eviction and their replacement by non-histone DNA-packaging proteins. Although the occurrence of this histone hyperacetylation has been correlated with histone removal for a long time, the underlying mechanisms have remained largely obscure. Important recent discoveries have not only shed light on how histone acetylation could drive a subsequent transformation in genome organization but also revealed that the associated nucleosome dismantlement is a multi-step process, requiring the contribution of histone variants, critical destabilizing histone modifications and chromatin readers, including Brdt, working together to achieve the full packaging of the male genome, indispensable for the propagation of life.