Advanced Concentration

Genetics

Mentor

Daniel J. Driscoll, Ph.D., M.D.

Department

Molecular Genetics & Microbiology

Enrolled 

August 2000

Lab Room

RG-277

Lab Phone

392-4101

E-mail

akhalil@ufl.edu

Education

B.A., Biology, University of Kansas

Dissertation Research

Histone modifications and chromatin dynamics of the mammalian inactive sex chromosomes

Abstract 1
      Based on the formation of the XY body at pachytene and expression studies of few X linked genes, the X and Y chromosomes seem to undergo transcriptional inactivation during mammalian spermatogenesis. However, the extent and the mechanism of X and Y inactivation (XYi) are not known. Further, unlike X inactivation in females, Xist expression and DNA methylation are not required.
      Here, we show that both the X and Y chromosomes begin at the pachytene stage to undergo a specific sequence of changes in their histone modifications. The changes which we observe are usually associated with transcriptional inactivation in somatic cells, and they coincide with the exclusion of RNA Polymerase II (H5) from the XY body. The X and Y chromosomes undergo extensive deacetylation at histone H4 and H3, and (di)methylation of lysine (K) 9 on histone 3, however, no changes in H3-K4 methylation. These changes persist even when the XY body disappears in late pachytene, and the X and Y chromosomes segregate from one another after the first meiotic division. However, by the spermatid stage, histone modifications of the X and Y revert back to those of active chromatin and RNA polymerase II (H5) reengages with both chromosomes. Our observations indicate that X and Y inactivation is extensive and persists even when the X and Y are separated in secondary spermatocytes.
      These novel findings 1) provide insights into epigenetic programming and chromatin dynamics during spermatogenesis; 2) show that the sequence of changes in histone modifications associated with X and Y inactivation during spermatogenesis has key differences from those associated with X inactivation (Xi) in female somatic cells, suggesting that the male germ line has developed a different strategy of Xi than the female somatic lineage.

Abstract 2
      It is now estimated that 150-200 genes escape X inactivation (Xi) in somatic cells of human females. These genes are clustered in several discrete regions on the X chromosome. However, it is not currently known how these genes escape Xi. Here, we show that although the human female inactive X chromosome is largely devoid of histone 3 lysine 4 (H3-K4) trimethylation, regions that escape Xi are enriched with this modification. Also, H3-K4 trimethylation, unlike histone acetylation, is restricted to discrete regions on metaphase chromosomes, and is completely absent from constitutive heterochromatin. In contrast to humans, there are only few genes that are known to escape Xi in the mouse. Therefore, we examined mouse female somatic cells with H3-K4 trimethylation to identify candidate regions with genes that escape Xi. We found the mouse female inactive X to have 7 discrete regions, including the pseudoautosomal region, that are enriched with H3-K4 trimethylation.
      Xi also occurs in males during spermatogenesis. We found the inactive X in mouse male meiosis to become devoid of H3-K4 trimethylation except for 7 discrete regions. Those regions are at the same chromosomal position as the ones we observed on the inactive X in somatic cells of the mouse female, suggesting that regions that escape Xi in females are either 1) marked during meiosis and/or 2) genes that escape Xi in females also escape Xi in male meiosis. To determine if there are regions that escape Xi during spermatogenesis, we examined male germ cells with an antibody for RNA polymerase II. Although RNA polymerase II is largely excluded from the inactive X in male meiosis, there are several discrete regions on the X and Y chromosomes that remain associated with RNA polymerase II, indicating the presence of genes that escape X and Y inactivation.
      In summary, 1) we have identified H3-K4 trimethylation as a cytogenetic mark for regions that escape Xi in females, 2) our results suggest that there are more genes that escape Xi in the mouse female, and 2) we have shown that there are regions that escape X and Y inactivation during spermatogenesis, those regions are also enriched with H3-K4 trimethytlation.

Publications

Khalil AM, Boyar FZ, Driscoll DJ.  Dynamic histone modifications mark sex chromosome inactivation and reactivation during mammalian spermatogenesis. Proc Natl Acad Sci U S A. 2004 Nov 9;101:16583-16587.  abstract

Khalil AM, Driscoll DJ.  Tri-methylation of Histone H3 Lysine 4 is an Epigenetic Mark for Regions Escaping X Inactivation.  (in preparation)

Khalil AM, Driscoll DJ.  Histone 3 Lysine 4 Dimethylation is Enriched on the Inactive X in Male Meiosis but Absent on the Inactive X in Females.  (in preparation)

Abstracts Presented at Meetings

Honors & Awards