Histone Retention Research Articles

Abnormal Gene Expression in Mouse Embryos Resulting from Excessive Sperm Histone Retention Due to Perturbed Poly(ADP-ribose) Metabolism During Spermiogenesis.

Abnormal Gene Expression in Mouse Embryos Resulting from Excessive Sperm Histone Retention Due to Perturbed Poly(ADP-ribose) Metabolism During Spermiogenesis.

Genome-wide analysis identifies changes in histone retention and epigenetic modifications at developmental and imprinted gene loci in the sperm of infertile men.

The sperm chromatin of fertile men retains a small number of nucleosomes that are enriched at developmental gene promoters and imprinted gene loci. This unique chromatin packaging at certain gene promoters provides these genomic loci the ability to convey instructive epigenetic information to the zygote, potentially expanding the role and significance of the sperm epigenome in embryogenesis. We hypothesize that changes in chromatin packaging may be associated with poor reproductive outcome. Seven patients with reproductive dysfunction were recruited: three had unexplained poor embryogenesis during IVF and four were diagnosed with male infertility and previously shown to have altered protamination. Genome-wide analysis of the location of histones and histone modifications was analyzed by isolation and purification of DNA bound to histones and protamines. The histone-bound fraction of DNA was analyzed using high-throughput sequencing, both initially and following chromatin immunoprecipitation. The protamine-bound fraction was hybridized to agilent arrays. DNA methylation was examined using bisulfite sequencing. Unlike fertile men, five of seven infertile men had non-programmatic (randomly distributed) histone retention genome-wide. Interestingly, in contrast to the total histone pool, the localization of H3 Lysine 4 methylation (H3K4me) or H3 Lysine 27 methylation (H3K27me) was highly similar in the gametes of infertile men compared with fertile men. However, there was a reduction in the amount of H3K4me or H3K27me retained at developmental transcription factors and certain imprinted genes. Finally, the methylation status of candidate developmental promoters and imprinted loci were altered in a subset of the infertile men. This initial genome-wide analysis of epigenetic markings in the sperm of infertile men demonstrates differences in composition and epigenetic markings compared with fertile men, especially at certain imprinted and developmental loci. Although no single locus displays a complete change in chromatin packaging or DNA modification, the data suggest that moderate changes throughout the genome exist and may have a cumulative detrimental effect on fecundity.

2019 EFFECT OF MICROSURGICAL VARICOCELECTOMY ON HUMAN SPERM DNA INTEGRITY AND CHROMATIN COMPACTION

You have accessJournal of UrologyInfertility: Physiology, Pathophysiology, Basic Research1 Apr 20112019 EFFECT OF MICROSURGICAL VARICOCELECTOMY ON HUMAN SPERM DNA INTEGRITY AND CHROMATIN COMPACTION Naif Alhathal, Maria San Gabriel, and Armand Zini Naif AlhathalNaif Alhathal Montreal, Canada More articles by this author , Maria San GabrielMaria San Gabriel Montreal, Canada More articles by this author , and Armand ZiniArmand Zini Montreal, Canada More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.2247AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Human sperm DNA damage may adversely affect reproductive outcomes, and the spermatozoa of infertile men possess substantially more DNA damage than that of fertile men. Further to our previous study on the effect of varicocelectomy on sperm DNA integrity (DFI= DNA fragmentation index) using sperm chromatin structure assay (SCSA), the objective of this study was to further assess human sperm chromatin integrity before and after varicocelectomy by evaluating aniline blue staining (AB) in these samples. METHODS We evaluated 20 men who underwent microsurgical varicocelectomy for clinical varicocele and 6 controls (sperm donors) at our institution. We examined standard semen parameters, sperm DNA integrity (by SCSA) and chromatin integrity (by aniline blue stain) before and after varicocelectomy and for the control group. RESULTS The percentage of sperm with positive AB staining (histone retention) decreased significantly after surgery (from 13.5 to 5.4%). The %DFI (DNA fragmentation index) also decreased significantly (as previously shown). However, there were no significant relationship between AB staining and %DFI, motility or concentration. The only notable relationship was between AB staining and HDS (high DNA stainability–an index of chromatin compaction) post varicocelectomy. Furthermore, the percentage of sperm with positive AB staining was significantly less in the control compared to varicocele patients before surgery (2.5% vs 13.5%). The %DFI also was significantly less in the control group (7% vs 20%). CONCLUSIONS The data show that varicocelectomy is associated with a consistent improvement in sperm DNA integrity and chromatin compaction using two different assays of sperm chromatin integrity (SCSA, Aniline Blue). However, the poor relationship between SCSA parameters (e.g. %DFI) and AB staining suggests that these assays measure different aspects of sperm function. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e807 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Naif Alhathal Montreal, Canada More articles by this author Maria San Gabriel Montreal, Canada More articles by this author Armand Zini Montreal, Canada More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

Poly(ADP-Ribose) Polymerases PARP1 and PARP2 Modulate Topoisomerase II Beta (TOP2B) Function During Chromatin Condensation in Mouse Spermiogenesis1

To achieve the specialized nuclear structure in sperm necessary for fertilization, dramatic chromatin reorganization steps in developing spermatids are required where histones are largely replaced first by transition proteins and then by protamines. This entails the transient formation of DNA strand breaks to allow for, first, DNA relaxation and then chromatin compaction. However, the nature and origin of these breaks are not well understood. We previously reported that these DNA strand breaks trigger the activation of poly(ADP-ribose) (PAR) polymerases PARP1 and PARP2 and that interference with PARP activation causes poor chromatin integrity with abnormal retention of histones in mature sperm and impaired embryonic survival. Here we show that the activity of topoisomerase II beta (TOP2B), an enzyme involved in DNA strand break formation in elongating spermatids, is strongly inhibited by the activity of PARP1 and PARP2 in vitro, and this is in turn counteracted by the PAR-degrading activity of PAR glycohydrolase. Moreover, genetic and pharmacological PARP inhibition both lead to increased TOP2B activity in murine spermatids in vivo as measured by covalent binding of TOP2B to the DNA. In summary, the available data suggest a functional relationship between the DNA strand break-generating activity of TOP2B and the DNA strand break-dependent activation of PARP enzymes that in turn inhibit TOP2B. Because PARP activity also facilitates histone H1 linker removal and local chromatin decondensation, cycles of PAR formation and degradation may be necessary to coordinate TOP2B-dependent DNA relaxation with histone-to-protamine exchange necessary for spermatid chromatin remodeling.

The paternal epigenome and embryogenesis: poising mechanisms for development

The scope of paternal contributions during early embryonic development has long been considered limited. Dramatic changes in chromatin structure throughout spermatogenesis have been thought to leave the sperm void of complex layers of epigenetic regulation over the DNA blueprint, thus leaving the balance of that regulation to the oocyte. However, recent work in the fields of epigenetics and male factor infertility has placed this long-held, and now controversial dogma, in a new light. Elegant studies investigating chromatin and epigenetic modifications in the developing sperm cell have provided new insights that may establish a more critical role for the paternal epigenome in the developing embryo. DNA methylation, histone tail modifications, targeted histone retention and protamine incorporation into the chromatin have great influence in the developing sperm cell. Perturbations in the establishment and/or maintenance of any of these epigenetic marks have been demonstrated to affect fertility status, ranging in severity from mild to catastrophic. Sperm require this myriad of chromatin structural changes not only to serve a protective role to DNA throughout spermatogenesis and future delivery to the egg, but also, it appears, to contribute to the developmental program of the future embryo. This review will focus on our current understanding of the epigenetics of sperm. We will discuss sperm-specific chromatin modifications that result in genes essential to development being poised for activation early in embryonic development, the disruption of which may result in reduced fecundity.

Poly(ADP-ribose) Metabolism Is Essential for Proper Nucleoprotein Exchange During Mouse Spermiogenesis

Sperm chromatin is organized in a protamine-based, highly condensed form, which protects the paternal chromosome complement in transit, facilitates fertilization, and supports correct gene expression in the early embryo. Very few histones remain selectively associated with genes and defined regulatory sequences essential to embryonic development, while most of the genome becomes bound to protamine during spermiogenesis. Chromatin remodeling processes resulting in the dramatically different nuclear structure of sperm are poorly understood. This study shows that perturbation of poly(ADP-ribose) (PAR) metabolism, which is mediated by PAR polymerases and PAR glycohydrolase in response to naturally occurring endogenous DNA strand breaks during spermatogenesis, results in the abnormal retention of core histones and histone linker HIST1H1T (H1t) and H1-like linker protein HILS1 in mature sperm. Moreover, genetic or pharmacological alteration of PAR metabolism caused poor sperm chromatin quality and an abnormal nuclear structure in mice, thus reducing male fertility.

106. NUCLEOSOME RETENTION DURING CHROMATIN PACKAGING IN HUMAN SPERMATOZOA

In contrast to the histone packaging of somatic cells, spermatozoa are predominantly packaged by the protamine proteins. However, human spermatozoa retain ~15% histone packaging. Regions that are left nucleosome bound could be: either genes that are active shortly after fertilisation or genes that are transcribed late during spermatogenesis. DNA damage at histone bound loci would be of consequence to spermatogenesis and/or to a resulting embryo post-fertilisation. Western blot analysis confirmed the presence of acetylated H3 and H4 in sperm. Interestingly, we identified the presence of these modified histones in isolated good quality sperm considered to have complete packaging. This indicates that histone retention is not a consequence of aberrant chromatin remodelling. Using a combination of ChIP (chromatin immunoprecipitation) and tiling arrays we have identified regions bound by acetylated histone 3 (H3). ChIP-PCR confirmed histone retention at several of these loci. For the previously identified loci in exon 1 of the TNP-2 gene we were able to narrow the region bound to the size of one nucleosome only. Using a modified form of ChIP known as carrier ChIP we examined histone retention in individual ejaculates. We demonstrate that humans consistently retain acetylated H3 at the same loci. Retention of one nucleosome at the TNP-2 loci was maintained across ejaculates and donors. In conclusion, retention of acetylated H3 is consistently maintained during packaging of the genome in spermiogenesis.

Alteration of Poly(ADP-ribose) Metabolism Leads to Abnormal Nucleoprotein Exchange in Spermiogenesis of the Mouse.

Chromatin integrity has been identified as an important prerequisite for normal sperm cell function. The etiology of male idiopathic infertility associated with high sperm nuclear histone content and residual DNA strand breaks is not well understood. Most likely this type of sperm cell defect will be caused by faulty or incomplete execution of chromatin remodeling steps and insufficient DNA strand break management during spermatid development. Based on earlier observations, we hypothesized that poly(ADP-ribose) (PAR) metabolism is required for chromatin remodeling steps involving DNA strand breaks during spermatid nuclear elongation. At least two members of the multifunctional family of poly(ADP-ribose) polymerases, PARP1 and PARP2, are intimately involved in DNA repair where their enzymatic activity is directly triggered by DNA strand breaks to produce PAR by cleavage of NAD. Only one enzyme is capable of rapid degradation of the unique biopolymer, poly(ADP-ribose) glycohydrolase (PARG) which exists in four isoforms encoded by alternatively spliced transcripts from a single gene. Using Parp1-/-, Parg(110)-/-, and double gene disrupted mice, which all have different forms of abnormal poly(ADP-ribose) metabolism, we showed that nuclear shaping and DNA integrity are compromised by modulation of this pathway. In the present study we investigated the exchange of histones and transition proteins by protamines in a similar panel of gene disrupted mice and by pharmacological targeting using PJ34 as a small molecule competitive inhibitor of PARP1 and PARP2. Abnormal degrees of histone retention in spermatozoa, associated with varying degrees of subfertility in these mouse strains support the view that PAR metabolism is carefully regulated during spermiogenesis and that pharmacological or genetic manipulation of both, PARP1 and PARP2 will lead to compromised sperm chromatin quality similar to that observed in clinical cases of increased sperm histone levels. This work has been supported by NIH HD048837 (RGM) and in part by intramural funding through the Mari Lowe Center for Comparative Oncology, Philadelphia. (platform)

Protamine ratio and the level of histone retention in sperm selected from a density gradient preparation

Fertile males express two forms of sperm nuclear proteins, protamine 1 (P1) and protamine 2 (P2), in roughly equal quantities, whereas some infertile men have been shown to have a reduction in protamine content and an increase in the level of histones retained in mature sperm. In this study, we assessed histone and protamine levels in spermatozoa isolated from different layers of a density gradient centrifugation column to evaluate the nuclear protein content of the sperm population selected. Protamine levels were measured using acid gel electrophoresis and immunofluorescence, and the percentage of cells retaining histones was evaluated using aniline staining and immunofluorescence. Our data suggests that there is an inverse correlation between P1/P2 ratio and the level of histone expression in the different layers of the density gradient. Paradoxically, the 90% layer had a lower P1/P2 ratio, which corresponded with an increase in histone expression. It is concluded that although the sperm population selected in the 90% layer of the density gradient columns had a lower P1/P2 ratio, it was yet similar to the P1/P2 ratio observed in previously screened fertile donors.

Histone- and protamine-DNA association: conservation of different patterns within the beta-globin domain in human sperm.

Most DNA in human sperm is bound to highly basic proteins called protamines, but a small proportion is complexed with histones similar to those found in active chromatin. This raises the intriguing possibility that histones in sperm are marking sets of genes that will be preferentially activated during early development. We have examined the chromatin structure of members of the beta-globin gene family, which are expressed at different times in development, and the protamine 2 gene, which is expressed in spermatids prior to the widespread displacement of histones by transition proteins. The genes coding for epsilon and gamma globin, which are active in the embryonic yolk sac, contain regions which are histone associated in the sperm. No histone-associated regions are present at the sites tested within the beta- and delta-globin genes which are silent in the embryonic yolk sac. The trends of histone or protamine association are consistent for samples from the same person, and no significant between-subject variations in these trends are found for 13 of the 15 fragments analyzed in the two donors. The results suggest that sperm chromatin structures are generally similar in different men but that the length of the histone-associated regions can vary. The association of sperm DNA with histones or protamines sometimes changes within as little as 400 bp of DNA, suggesting that there is fine control over the retention of histones.

Remodeling chromatin structures for transcription: what happens to the histones?

Activation of gene transcription in vivo is accompanied by an alteration of chromatin structure. The specific binding of transcriptional activators disrupts nucleosomal arrays, suggesting that the primary steps leading to transcriptional initiation involve interactions between activators and chromatin. The affinity of transcription factors for nucleosomal DNA is determined by the location of recognition sequences within nucleosomes, and by the cooperative interactions of multiple proteins targeting binding sites contained within the same nucleosomes. In addition, two distinct types of enzymatic complexes facilitate binding of transcription factors to nucleosomal DNA. These include type A histone acetyltransferases (e.g. GCN5/ADA transcriptional adaptor complex) and ATP-driven molecular machines that disrupt histone-DNA interactions (e.g. SWI/SNF and NURF complexes). These observations raise the important question of what happens to the histones during chromatin remodeling. We discuss evidence supporting the retention of histones at transcription factor-bound sequences as well as two alternative pathways of histone loss from gene control elements upon transcription factor binding: histone octamer sliding and histone dissociation.

Dynamics of Streptococcus Histone Retention by Mouse Kidneys

To study the interaction of streptococcal histone-like protein with renal tissue, 2 groups of 12 mice were injected intravenously with either radioiodinated histone or bovine serum albumin. At intervals over 48 hr, 2 mice from each group were anesthetized and perfused with tissue culture medium and the amounts of radioactivity were measured in blood, urine, and kidneys. The streptococcal protein rapidly disappeared from the blood and accumulated in renal tissue. Kidney radioactivity was maximal at 2 hr and then declined steadily over the ensuing 46 hr. Retention of streptococcal protein in renal tissue was 2 orders higher than that of BSA throughout the experiment. Immunofluorescence staining of kidney sections showed that the histone protein was adsorbed to the basement membranes of the glomeruli and collecting tubules. There were similar rates of excretion of radioactivity in urine by the two groups of mice. Injection of preformed complexes of streptococcal histone and rabbit antibodies into a third group of mice resulted in deposits of immune complexes in glomeruli but did not change the overall rate at which the radiolabeled streptococcal protein was distributed. The accumulation of streptococcal histone in renal tissue, independently of antibodies or while contained in circulating immune complexes, makes it a potential virulence factor in the pathogenesis of poststreptococcal glomerulonephritis. Its pathogenic properties remain to be studied in an appropriate animal model.

Histones from exponential and stationary L-cells: Evidence for differential binding of lysine-rich and arginine-rich fractions in chromatin

Histones from exponential and stationary-phase mouse L-cells were quantitated after acrylamide gel electrophoresis in order to investigate cell cycle-dependent changes in the mode of binding of the various fractions in chromatin. By introducing various concentrations of citrate and divalent cations in the medium used for cell lysis and isolation of nuclei prior to histone extraction it was possible to demonstrate that certain histone fractions are preferentially retained in either exponential or stationary-phase nuclei. Differential retention of lysine-rich F 1 was most evident when the lysing medium contained 1 m m Mg 2+ and Ca 2+ and 5 m m citrate (pH 2.75). In these conditions twice as much F 1 is retained in stationary as in exponential nuclei. Differential retention of arginine-rich histones was most evident when the lysing medium contained 10 m m Mg 2+ and Ca 2+ and no citrate. In these conditions more F 2a 1 is retained in exponential than in stationary nuclei while the opposite is true for F 3. However, the total amount of arginine-rich fractions (F 2a 1 + F 3) retained was found to be the same in both cell phases. The results are discussed in relation to known structural features of the histones.