Judd C. Rice

PR-Set7 Is a Nucleosome-Specific Methyltransferase that Modifies Lysine 20 of Histone H4 and Is Associated with Silent Chromatin

We have purified a human histone H4 lysine 20 methyltransferase and cloned the encoding gene, PR/SET07. A mutation in Drosophila pr-set7 is lethal: second instar larval death coincides with the loss of H4 lysine 20 methylation, indicating a fundamental role for PR-Set7 in development. Transcriptionally competent regions lack H4 lysine 20 methylation, but the modification coincided with condensed chromosomal regions on polytene chromosomes, including chromocenter and euchromatic arms. The Drosophila male X chromosome, which is hyperacetylated at H4 lysine 16, has significantly decreased levels of lysine 20 methylation compared to that of females. In vitro, methylation of lysine 20 and acetylation of lysine 16 on the H4 tail are competitive. Taken together, these results support the hypothesis that methylation of H4 lysine 20 maintains silent chromatin, in part, by precluding neighboring acetylation on the H4 tail.

Code of silence.

Using genetic approaches and a filamentous fungus, molecular biologists have found further evidence of an intimate relationship between genomes and the histone proteins that evolved to package them.

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.

Dynamic regulation of the PR-Set7 histone methyltransferase is required for normal cell cycle progression

Although the PR-Set7/Set8/KMT5a histone H4 Lys 20 monomethyltransferase (H4K20me1) plays an essential role in mammalian cell cycle progression, especially during G2/M, it remained unknown how PR-Set7 itself was regulated. In this study, we discovered the mechanisms that govern the dynamic regulation of PR-Set7 during mitosis, and that perturbation of these pathways results in defective mitotic progression. First, we found that PR-Set7 is phosphorylated at Ser 29 (S29) specifically by the cyclin-dependent kinase 1 (cdk1)/cyclinB complex, primarily from prophase through early anaphase, subsequent to global accumulation of H4K20me1. While S29 phosphorylation did not affect PR-Set7 methyltransferase activity, this event resulted in the removal of PR-Set7 from mitotic chromosomes. S29 phosphorylation also functions to stabilize PR-Set7 by directly inhibiting its interaction with the anaphase-promoting complex (APC), an E3 ubiquitin ligase. The dephosphorylation of S29 during late mitosis by the Cdc14 phosphatases was required for APC(cdh1)-mediated ubiquitination of PR-Set7 and subsequent proteolysis. This event is important for proper mitotic progression, as constitutive phosphorylation of PR-Set7 resulted in a substantial delay between metaphase and anaphase. Collectively, we elucidated the molecular mechanisms that control PR-Set7 protein levels during mitosis, and demonstrated that its orchestrated regulation is important for normal mitotic progression.

Hormone therapy attenuates exercise-induced skeletal muscle damage in postmenopausal women

Hormone therapy (HT) is a potential treatment to relieve symptoms of menopause and prevent the onset of disease such as osteoporosis in postmenopausal women. We evaluated changes in markers of exercise-induced skeletal muscle damage and inflammation [serum creatine kinase (CK), serum lactate dehydrogenase (LDH), and skeletal muscle mRNA expression of IL-6, IL-8, IL-15, and TNF-alpha] in postmenopausal women after a high-intensity resistance exercise bout. Fourteen postmenopausal women were divided into two groups: women not using HT (control; n = 6, 59 +/- 4 yr, 63 +/- 17 kg) and women using traditional HT (HT; n = 8, 59 +/- 4 yr, 89 +/- 24 kg). Both groups performed 10 sets of 10 maximal eccentric repetitions of single-leg extension on the Cybex dynamometer at 60 degrees /s with 20-s rest periods between sets. Muscle biopsies of the vastus lateralis were obtained from the exercised leg at baseline and 4 h after the exercise bout. Gene expression was determined by RT-PCR for IL-6, IL-8, IL-15, and TNF-alpha. Blood draws were performed at baseline and 3 days after exercise to measure CK and LDH. Independent t-tests were performed to test group differences (control vs. HT). A probability level of P <or= 0.05 was used to determine statistical significance. We observed significantly greater changes in mRNA expression of IL-6, IL-8, IL-15, and TNF-alpha (P <or= 0.01) in the control group compared with the HT group after the exercise bout. CK and LDH levels were significantly greater after exercise (P <or= 0.01) in the control group. Postmenopausal women not using HT experienced greater muscle damage after maximal eccentric exercise, indicating a possible protective effect of HT against exercise-induced skeletal muscle damage.

Role for the nuclear receptor-binding SET domain protein 1 (NSD1) methyltransferase in coordinating lysine 36 methylation at histone 3 with RNA polymerase II function

The NSD (nuclear receptor-binding SET domain protein) family encodes methyltransferases that are important in multiple aspects of development and disease. Perturbations in NSD family members can lead to Sotos syndrome and Wolf–Hirschhorn syndrome as well as cancers such as acute myeloid leukemia. Previous studies have implicated NSD1 (KMT3B) in transcription and methylation of histone H3 at lysine 36 (H3-K36), but its molecular mechanism in these processes remains largely unknown. Here we describe an NSD1 regulatory network in human cells. We show that NSD1 binds near various promoter elements and regulates multiple genes that appear to have a concerted role in various processes, such as cell growth/cancer, keratin biology, and bone morphogenesis. In particular, we show that NSD1 binding is concentrated upstream of gene targets such as the bone morphogenetic protein 4 (BMP4) and zinc finger protein 36 C3H type-like 1 (ZFP36L1/TPP). NSD1 regulates the levels of the various forms of methylation at H3-K36 primarily, but not exclusively, within the promoter proximal region occupied by NSD1. At BMP4 we find that this reduces the levels of RNAP II recruited to the promoter, suggesting a role for NSD1-dependent methylation in initiation. Interestingly, we also observe that the RNAP II molecules that lie within BMP4 have inappropriate persistence of serine-5 phosphorylation and reduced levels of serine-2 phosphorylation within the C-terminal domain (CTD) of the large subunit of RNAP II. Our findings indicate that NSD1 regulates RNAP II recruitment to BMP4, and failure to do so leads to reduced gene expression and abrogated levels of H3K36Me and CTD phosphorylation.

PR‐Set7‐mediated monomethylation of histone H4 lysine 20 at specific genomic regions induces transcriptional repression

Increasing evidence indicates that the post‐translational modifications of the histone proteins play critical roles in all eukaryotic DNA‐templated processes. To gain further biological insights into two of these modifications, the mono‐ and trimethylation of histone H4 lysine 20 (H4K20me1 and H4K20me3), ChIP‐chip experiments were performed to identify the precise genomic regions on human chromosomes 21 and 22 occupied by these two modifications. Detailed analysis revealed that H4K20me1 was preferentially enriched within specific genes; most significantly between the first ∼5% and 20% of gene bodies. In contrast, H4K20me3 was preferentially targeted to repetitive elements. Among genes enriched in H4K20me3, the modification was typically targeted to a small region ∼1 kb upstream of transcription start. Our collective findings strongly suggest that H4K20me1 and H4K20me3 are both physically and functionally distinct. We next sought to determine the role of H4K20me1 in transcription since this has been controversial. Following the reduction of PR‐Set7/Set8/KMT5a and H4K20me1 in cells by RNAi, all H4K20me1‐associated genes analyzed displayed an ∼2‐fold increase in gene expression; H4K20me3‐associated genes displayed no changes. Similar results were obtained using a catalytically dead dominant negative PR‐Set7 indicating that H4K20me1, itself, is essential for the selective transcriptional repression of H4K20me1‐associated genes. Furthermore, we determined that the H4K20me1‐associated DNA sequences were sufficient to nucleate H4K20me1 and induce repression in vivo. Our findings reveal the molecular mechanisms of a mammalian transcriptional repressive pathway whereby the DNA sequences within specific gene bodies are sufficient to nucleate the monomethylation of H4K20 which, in turn, reduces gene expression by half. J. Cell. Biochem. 110: 609–619, 2010.

Influence of hormone replacement therapy on eccentric exercise induced myogenic gene expression in postmenopausal women

Hormone replacement therapy (HRT) is used in postmenopausal women to relieve symptoms of menopause and prevent osteoporosis. We sought to evaluate changes in mRNA expression of key myogenic factors in postmenopausal women taking and not taking HRT following a high-intensity eccentric resistance exercise. Fourteen postmenopausal women were studied and included 6 control women not using HRT (59 +/- 4 years, 63 +/- 17 kg) and 8 women using traditional HRT (59 +/- 4 yr, 89 +/- 24 kg). Both groups performed 10 sets of 10 maximal eccentric repetitions of single-leg extension on a Cybex dynamometer at 60 degrees /s. Muscle biopsies of the vastus lateralis were obtained from the exercised leg at baseline and 4 h after the exercise bout. Gene expression was determined using RT-PCR for follistatin, forkhead box 3A (FOXO3A), muscle atrophy F-box (MAFbx), muscle ring finger-1 (MuRF-1), myogenic differentiation factor (MyoD), myogenin, myostatin, myogenic factor 5 (Myf5), and muscle regulatory factor 4 (MRF4). At rest, the HRT group expressed higher levels of MyoD, myogenin, Myf5, MRF4, and follistatin (P < 0.05). In response to eccentric exercise, follistatin, MyoD, myogenin, Myf5, and MRF4 were significantly increased (P <or= 0.05) and FOXO3A, MAFbx, MuRF-1, and myostatin were significantly decreased in the control and HRT groups (P <or= 0.05). Significantly greater changes in mRNA expression of follistatin, FOXO3A, MAFbx, MuRF-1, MyoD, myogenin, myostatin, Myf5, and MRF4 (p<or=0.05) occurred in the HRT group than in the control group after exercise. These data suggest that postmenopausal women using HRT express higher myogenic regulatory factor gene expression, which may reflect an attempt to preserve muscle mass. Furthermore, postmenopausal women using HRT experienced a greater myogenic response to maximal eccentric exercise.

Human SFMBT is a transcriptional repressor protein that selectively binds the N-terminal tail of histone H3

Human SFMBT (hSFMBT) is postulated to be a Polycomb (PcG) protein. Similar to other PcG proteins, we found that hSFMBT displays robust transcriptional repressor activity. In addition, hSFMBT localized to the nucleus where it strongly associates with chromatin by directly and selectively binding the N‐terminal tail of histone H3. Importantly, we discovered that the four tandem MBT repeats of hSFMBT were sufficient for nuclear matrix‐association, N‐terminal tail H3 binding, and required for transcriptional repression. These findings indicate that the tandem MBT repeats form a functional structure required for biological activity of hSFMBT and predict similar properties for other MBT domain‐containing proteins.