Jennifer K. Sims

Catalytic Function of the PR-Set7 Histone H4 Lysine 20 Monomethyltransferase Is Essential for Mitotic Entry and Genomic Stability

Histone-modifying enzymes play a critical role in modulating chromatin dynamics. In this report we demonstrate that one of these enzymes, PR-Set7, and its corresponding histone modification, the monomethylation of histone H4 lysine 20 (H4K20), display a distinct cell cycle profile in mammalian cells: low at G1, increased during late S phase and G2, and maximal from prometaphase to anaphase. The lack of PR-Set7 and monomethylated H4K20 resulted in a number of aberrant phenotypes in several different mammalian cell types. These include the inability of cells to progress past G2, global chromosome condensation failure, aberrant centrosome amplification, and substantial DNA damage. By employing a catalytically dead dominant negative PR-Set7 mutant, we discovered that its mono-methyltransferase activity was required to prevent these phenotypes. Importantly, we demonstrate that all of the aberrant phenotypes associated with the loss of PR-Set7 enzymatic function occur independently of p53. Collectively, our findings demonstrate that PR-Set7 enzymatic activity is essential for mammalian cell cycle progression and for the maintenance of genomic stability, most likely by monomethylating histone H4K20. Our results predict that alterations of this pathway could result in gross chromosomal aberrations and aneuploidy.

A trans-tail histone code defined by monomethylated H4 Lys-20 and H3 Lys-9 demarcates distinct regions of silent chromatin

The specific post-translational modifications of the histone proteins are associated with specific DNA-templated processes, such as transcriptional activation or repression. To investigate the biological role(s) of histone H4 lysine 20 (H4 Lys-20) methylation, we created a novel panel of antibodies that specifically detected mono-, di-, or trimethylated H4 Lys-20. We report that the different methylated forms of H4 Lys-20 are compartmentalized within visually distinct, transcriptionally silent regions in the mammalian nucleus. Interestingly, direct comparison of methylated H4 Lys-20 with the different methylated states of histone H3 lysine 9 (H3 Lys-9) revealed significant overlap and exclusion between the specific groups of methyl modifications. Trimethylated H4 Lys-20 and H3 Lys-9 were both selectively enriched within pericentric heterochromatin. Similarly, monomethylated H4 Lys-20 and H3 Lys-9 partitioned together and the dimethylated forms partitioned together within the chromosome arms; however, the mono- and dimethylated modifications were virtually exclusive. These findings strongly suggest that the combinatorial presence or absence of the different methylated states of H4 Lys-20 and H3 Lys-9 define particular types of silent chromatin. Consistent with this, detailed analysis of monomethylated H4 Lys-20 and H3 Lys-9 revealed that both were preferentially and selectively enriched within the same nucleosome particle in vivo. Collectively, these findings define a novel trans-tail histone code involving monomethylated H4 Lys-20 and H3 Lys-9 that act cooperatively to mark distinct regions of silent chromatin within the mammalian epigenome.

PR-Set7 Establishes a Repressive trans-Tail Histone Code That Regulates Differentiation

ABSTRACT Posttranslational modifications of the DNA-associated histone proteins play fundamental roles in eukaryotic transcriptional regulation. We previously discovered a novel trans-tail histone code involving monomethylated histone H4 lysine 20 (H4K20) and H3 lysine 9 (H3K9); however, the mechanisms that establish this code and its function in transcription were unknown. In this report, we demonstrate that H3K9 monomethylation is dependent upon the PR-Set7 H4K20 monomethyltransferase but independent of its catalytic function, indicating that PR-Set7 recruits an H3K9 monomethyltransferase to establish the trans-tail histone code. We determined that this histone code is involved in a transcriptional regulatory pathway in vivo whereby monomethylated H4K20 binds the L3MBTL1 repressor protein to repress specific genes, including RUNX1, a critical regulator of hematopoietic differentiation. The selective loss of monomethylated H4K20 at the RUNX1 promoter resulted in the displacement of L3MBTL1 and a concomitant increase in RUNX1 transcription. Importantly, the lack of monomethylated H4K20 in the human K562 multipotent cell line was specifically associated with spontaneous megakaryocytic differentiation, in part, by activating RUNX1. Our findings demonstrate that this newly described repression pathway is required for regulating proper megakaryopoiesis and suggests that it is likely to function similarly in other multipotent cell types to regulate specific differentiation pathways.

Exercise Does Not Influence Myostatin and Follistatin Messenger RNA Expression in Young Women

Jensky, NE, Sims, JK, Dieli-Conwright, CM, Sattler, FR, Rice, JC, and Schroeder, ET. Exercise does not influence myostatin and follistatin messenger RNA expression in young women. J Strength Cond Res 24(2): 522-530, 2010-We evaluated changes in myostatin, follistatin, and MyoD messenger RNA (mRNA) gene expression using eccentric exercise (EE) and concentric exercise (CE) as probes to better understand the mechanisms of muscle hypertrophy in young women. Twelve women performed single-leg maximal eccentric (n = 6, 25 ± 1 years, 59 ± 7 kg) or concentric (n = 6, 24 ± 1 years, 65 ± 7 kg) isokinetic knee extension exercise for 7 sessions. Muscle biopsies were taken from the vastus lateralis at baseline, 8 hours after the first exercise session, and 8 hours after the seventh exercise session. In the EE group, there were no changes in myostatin and follistatin (p ≥ 0.17); however, MyoD expression increased after 1 exercise bout (p = 0.02). In the CE group, there were no changes in myostatin, follistatin, or MyoD mRNA gene expression (p ≥ 0.07). Differences between the EE and CE groups were not significant (p ≥ 0.05). These data suggest that a single bout or multiple bouts of maximal EE or CE may not significantly alter myostatin or follistatin mRNA gene expression in young women. However, MyoD mRNA expression seems to increase only after EE.

The PR-Set7 binding domain of Riz1 is required for the H4K20me1-H3K9me1 trans-tail ‘histone code’ and Riz1 tumor suppressor function

PR-Set7/Set8/KMT5a is the sole histone H4 lysine 20 monomethyltransferase (H4K20me1) in metazoans and is essential for proper cell division and genomic stability. We unexpectedly discovered that normal cellular levels of monomethylated histone H3 lysine 9 (H3K9me1) were also dependent on PR-Set7, but independent of its catalytic activity. This observation suggested that PR-Set7 interacts with an H3K9 monomethyltransferase to establish the previously reported H4K20me1-H3K9me1 trans-tail ‘histone code’. Here we show that PR-Set7 specifically and directly binds the C-terminus of the Riz1/PRDM2/KMT8 tumor suppressor and demonstrate that the N-terminal PR/SET domain of Riz1 preferentially monomethylates H3K9. The PR-Set7 binding domain was required for Riz1 nuclear localization and maintenance of the H4K20me1-H3K9me1 trans-tail ‘histone code’. Although Riz1 can function as a repressor, Riz1/H3K9me1 was dispensable for the repression of genes regulated by PR-Set7/H4K20me1. Frameshift mutations resulting in a truncated Riz1 incapable of binding PR-Set7 occur frequently in various aggressive cancers. In these cancer cells, expression of wild-type Riz1 restored tumor suppression by decreasing proliferation and increasing apoptosis. These phenotypes were not observed in cells expressing either the Riz1 PR/SET domain or PR-Set7 binding domain indicating that Riz1 methyltransferase activity and PR-Set7 binding domain are both essential for Riz1 tumor suppressor function.