Transcription Silencing and Chromatin

Eukaryotic gene expression is greatly influenced by chromatin structure. In general, productive transcription is associated with open, accessible genomic regions, whereas genes located in closely packed genomic regions are transcriptionally repressed. Many recent studies show that post-translational modifications of histones are crucial for the specification of chromatin structure, which has profound implications in epigenetic control of gene expression. We are interested in understanding the molecular mechanisms of chromatin modification and remodeling in eukaryotic gene expression. The following is a list of our representative studies in the past few years in this area. Recognition of histone H3 Lysine 4 by the double tudor domain Recognition of histone H3 Lysine 27 by the drosophila polycomb protein Deciphering NAD-dependent deacetylases. SET domain histone lysine 9 methyltransferase Clr4 non-SET domain histone lysine 79 methyltransferase hDot1

mRNA Processing

Pre-mRNA splicing involves the ordered assembly of many components, including small nuclear ribonucleoprotein particles (snRNPs) and additional protein splicing factors. The fully assembled splicing machine is a large, 50-60S ribonucleoprotein complex known as the spliceosome. While crystallographic studies of the spliceosome and snRNPs have so far met with practical difficulties, we have focused on selected splicing factors that are important for splice-site selection and spliceosome assembly.

Recognition of histone H3 Lysine 4 by the double tudor domain

The double tudor domain of JMJD2A, a Jmjc domain-containing histone demethylase, binds methylated histone H3-K4 and H4-K20. We found that the double tudor domain has an interdigitated structure and the unusual fold is required for its ability to bind methylated histone tails. The cocrystal structure of the JMJD2A double tudor domain with a trimethylated H3-K4 peptide reveals that the trimethyl-K4 is bound in a cage of three aromatic residues, two of which are from the tudor-2 motif, while the binding specificity is determined by side-chain interactions involving amino acids from the tudor-1 motif. Our study provides mechanistic insights into recognition of methylated histone tails by tudor domains and reveals the structural intricacy of methyl-lysine recognition by two closely spaced effector domains. Huang Y, Fang J, Bedford MT, Zhang Y, Xu RM. Science. 2006 May 5; 312(5774):748-51. Back to Top

Recognition of histone H3 Lysine 27 by the drosophila polycomb protein

Much effort has been devoted to the analysis of histone methylation by various SET domain methyltransferases. However, our knowledge of the mechanism by which methylated histones are recognized is limited. The chromodomains of Polycomb and the heterochromatin associated protein HP1 bind the N-terminal tail of histone H3 methylated at lysine 27 and lysine 9, respectively. Nevertheless, the structural basis for binding specificity was not understood. We determined a 1.4 Å crystal structure of the Polycomb chromodomain in complex with a histone H3 peptide tri-methylated at lysine 27 (Min et al., 2003b). The structure revealed a conserved mode of methyl-lysine binding and identifies Polycomb-specific interactions with histone H3. The structure also allowed us to propose that a major determinant of Polycomb specificity resides in histone-histone interactions facilitated by a Polycomb chromodomain dimer. Min J, Zhang Y, Xu RM. Genes Dev. 2003 Aug 1;17(15):1823-8. Back to Top

Deciphering NAD-dependent deacetylases.

Transcriptional silencing in the budding yeast S. cerevisiae requires silencing information regulator (SIR) proteins. SIR2 is a novel NAD-dependent histone deacetylase that is important for transcriptional silencing, DNA repair, and life-span extension in S. cerevisiae. We determined the structure of an archaeal homolog of SIR2 in complex with NAD. This very first structure of a NAD-dependent deacetylase revealed that the protein consists of a large domain having a Rossmann fold and a small domain containing a three-stranded zinc ribbon motif. NAD is bound in a pocket between the two domains and in an inverted orientation, compared to the binding of NAD to other Rossmann-fold enzymes. The structure identified the active sites of NAD cleavage, histone deacetylation, and the binding site of acetyl-lysine. The structure allowed us to propose a detailed catalytic mechanism for this novel family of enzymes, which has been largely confirmed by several groups in subsequent studies. Min J, Landry J, Sternglanz R, Xu RM. Cell. 2001 Apr 20;105(2):269-79. Back to Top

SET domain histone lysine 9 methyltransferase Clr4

The SET domain-containing Clr4 protein, a close relative of Su(var)3-9 proteins in higher eukaryotes, specifically methylates lysine 9 of histone H3 and is essential for silencing in the fission yeast S. pombe. We determined the crystal structure of the catalytic domain of Clr4 (Min et al., 2002). The crystal structure revealed that the SET domain had an overall fold rich in b-strands. The active site consists of a S-adenosyl-l-methionine binding pocket and a connected groove that could accommodate the binding of the N-terminal tail of histone H3. The cysteine-rich pre-SET motif contains a triangular zinc cluster coordinated by nine cysteines distant from the active site, whereas the post-SET motif is flexible but close to the active site. Our structural analysis suggests that the post-SET motif will fold back, via the binding of another zinc ion coordinated by three cysteines in the post-SET motif and another cysteine in the SET domain, to form an enclosed catalytic pocket. This structure-based hypothesis was directly confirmed in a subsequent study of a separate SET domain protein. Min J, Zhang X, Cheng X, Grewal SI, Xu RM. Nat Struct Biol. 2002 Nov;9(11):828-32. Back to Top

non-SET domain histone lysine 79 methyltransferase hDot1

All SET domain histone methyltransferases known to date methylate lysine residues located at the flexible N-terminal histone tails. The yeast disruptor of telomeric (Dot) silencing protein Dot1 and its human homolog hDOT1L methylate lysine 79 of histone H3 in the core domain. Interestingly, Dot1 proteins appear to have a canonical methyltransferase fold common to DNA and arginine methyltransferases, which is rather curious as all previously known lysine methyltransferases have a SET domain fold. We have determined the structure of the catalytic domain of hDOT1L in complex with S-adenosyl-l-methionine (Min et al., 2003a). The structure revealed unique properties of hDOT1L that define its lysine specificity. Our structural and biochemical analyses provided mechanistic insights into the catalytic mechanism and nucleosomal specificity of the Dot1 family of unusual histone lysine methyltransferases. Min J, Feng Q, Li Z, Zhang Y, Xu RM. Cell. 2003 Mar 7;112(5):711-23. Back to Top

The interaction between silence information regulator 1 protein (Sir1p) and origin recognition complex 1 protein (Orc1p), the largest subunit of the origin recognition complex, plays an important role in the establishment of transcriptional silencing at the cryptic mating-type gene loci in Saccharomyces cerevisiae. Sir1p binds the N-terminal region of Orc1p encompassing a Bromo-adjacent homology (BAH) domain found in various chromatin-associated proteins. To understand the molecular mechanism of Sir protein recruitment, we have determined a 2.5-A cocrystal structure of the N-terminal domain of Orc1p in complex with the Orc1p-interacting domain of Sir1p. The structure reveals that Sir1p Orc1p-interacting domain has a bilobal structure: an alpha/beta N-terminal lobe and a C-terminal lobe resembling the Tudor domain royal family fold. The N-terminal lobe of Sir1p binds in a shallow groove between a helical subdomain and the BAH domain of Orc1p. The structure provides a mechanistic understanding of Orc1p-Sir1p interaction specificity, as well as insights into protein-protein interactions involving BAH domains in general. Hsu HC, Stillman B, Xu RM. Proc Natl Acad Sci USA. 2005 Jun 14;102(24):8519-24. Back to Top

The splicing history is imprinted on spliced mRNA through the deposition of a multiprotein complex, known as the exon junction complex (EJC), at ~20 nucleotides upstream of the exon-exon junction. Mago nashi (Mago) and Y14 are core components of the EJC, and they from a stable heterodimer that strongly associates with spliced mRNA. We determined a 1.85 Å structure of the Drosophila Mago-Y14 complex. Surprisingly, the structure showed that the canonical RNA binding surface of the Y14 RRM was involved in protein-protein interactions with Mago. This was the first observation that the RRM RNA binding surface could act as a protein-binding interface. This structure challenged the conventional wisdom of how the Mago-Y14 heterodimer might bind RNA and provided important insights into the molecular mechanisms of EJC assembly and RRM-mediated protein-protein interactions. Shi H, Xu RM. Genes Dev. 2003 Apr 15;17(8):971-6. Back to Top