Hugh W. Brock

MLL Targets SET Domain Methyltransferase Activity to Hox Gene Promoters

MLL, the human homolog of Drosophila trithorax, maintains Hox gene expression in mammalian embryos and is rearranged in human leukemias resulting in Hox gene deregulation. How MLL or MLL fusion proteins regulate gene expression remains obscure. We show that MLL regulates target Hox gene expression through direct binding to promoter sequences. We further show that the MLL SET domain is a histone H3 lysine 4-specific methyltransferase whose activity is stimulated with acetylated H3 peptides. This methylase activity is associated with Hox gene activation and H3 (Lys4) methylation at cis-regulatory sequences in vivo. A leukemogenic MLL fusion protein that activates Hox expression had no effect on histone methylation, suggesting a distinct mechanism for gene regulation by MLL and MLL fusion proteins.

Transcription of bxd Noncoding RNAs Promoted by Trithorax Represses Ubx in cis by Transcriptional Interference

Much of the genome is transcribed into long noncoding RNAs (ncRNAs). Previous data suggested that bithoraxoid (bxd) ncRNAs of the Drosophila bithorax complex (BX-C) prevent silencing of Ultrabithorax (Ubx) and recruit activating proteins of the trithorax group (trxG) to their maintenance elements (MEs). We found that, surprisingly, Ubx and several bxd ncRNAs are expressed in nonoverlapping patterns in both embryos and imaginal discs, suggesting that transcription of these ncRNAs is associated with repression, not activation, of Ubx. Our data rule out siRNA or miRNA-based mechanisms for repression by bxd ncRNAs. Rather, ncRNA transcription itself, acting in cis, represses Ubx. The Trithorax complex TAC1 binds the Ubx coding region in nuclei expressing Ubx, and the bxd region in nuclei not expressing Ubx. We propose that TAC1 promotes the mosaic pattern of Ubx expression by facilitating transcriptional elongation of bxd ncRNAs, which represses Ubx transcription.

TrxG and PcG Proteins but Not Methylated Histones Remain Associated with DNA through Replication

Propagation of gene-expression patterns through the cell cycle requires the existence of an epigenetic mark that re-establishes the chromatin architecture of the parental cell in the daughter cells. We devised assays to determine which potential epigenetic marks associate with epigenetic maintenance elements during DNA replication in Drosophila embryos. Histone H3 trimethylated at lysines 4 or 27 is present during transcription but, surprisingly, is replaced by nonmethylated H3 following DNA replication. Methylated H3 is detected on DNA only in nuclei not in S phase. In contrast, the TrxG and PcG proteins Trithorax and Enhancer-of-Zeste, which are H3K4 and H3K27 methylases, and Polycomb continuously associate with their response elements on the newly replicated DNA. We suggest that histone modification enzymes may re-establish the histone code on newly assembled unmethylated histones and thus may act as epigenetic marks.

Dimerization of MLL fusion proteins immortalizes hematopoietic cells

MLL fusion proteins are leukemogenic, but their mechanism is unclear. Induced dimerization of a truncated MLL immortalizes bone marrow and imposes a reversible block on myeloid differentiation associated with upregulation of Hox a7, a9, and Meis1. Both dimerized MLL and exon-duplicated MLL are potent transcriptional activators, suggesting a link between dimerization and partial tandem duplication of DNA binding domains of MLL. Dimerized MLL binds with higher affinity than undimerized MLL to a CpG island within the Hox a9 locus. However, MLL-AF9 is not dimerized in vivo. The data support a model in which either MLL dimerization/exon duplication or fusion to a transcriptional activator results in Hox gene upregulation and ultimately transformation.

Leukemogenic MLL Fusion Proteins Bind across a Broad Region of the Hox a9 Locus, Promoting Transcription and Multiple Histone Modifications

Chromosome translocations involving the mixed lineage leukemia gene MLL are associated with aggressive acute leukemias in both children and adults. Leukemogenic MLL fusion proteins delete the MLL SET domain Lys(4) methyltransferase activity and fuse MLL to 1 of >40 different translocation partners. Some MLL fusion proteins involve nuclear proteins that are transcriptional activators, whereas others have transcriptional activating activity but instead dimerize the truncated MLL molecule. Both types of MLL fusion proteins enforce persistent expression of Hox a9 and Meis1, which is pivotal for leukemogenesis through mechanisms that remain obscure. Here, we show that nuclear and dimerizable forms of MLL bind with a similar pattern to the Hox a9 locus that overlaps the distribution of wild-type MLL and deregulate transcription of three isoforms of Hox a9. Induction of MLL fusion protein activity is associated with increased levels of histone acetylation and Lys(4) methylation at Hox target genes. In addition, the MLL-ENL-ER protein, but not dimerized MLL, also induces dimethylation of histone H3 at Lys(79), suggesting alternative mechanisms for transcriptional activation.

A domain shared by the Polycomb group proteins Scm and ph mediates heterotypic and homotypic interactions.

The Sex comb on midleg (Scm) and polyhomeotic (ph) proteins are members of the Polycomb group (PcG) of transcriptional repressors. PcG proteins maintain differential patterns of homeotic gene expression during development in Drosophila flies. The Scm and ph proteins share a homology domain with 38% identity over a length of 65 amino acids, termed the SPM domain, that is located at their respective C termini. Using the yeast two-hybrid system and in vitro protein-binding assays, we show that the SPM domain mediates direct interaction between Scm and ph. Binding studies with isolated SPM domains from Scm and ph show that the domain is sufficient for these protein interactions. These studies also show that the Scm-ph and Scm-Scm domain interactions are much stronger than the ph-ph domain interaction, indicating that the isolated domain has intrinsic binding specificity determinants. Analysis of site-directed point mutations identifies residues that are important for SPM domain function. These binding properties, predicted alpha-helical secondary structure, and conservation of hydrophobic residues prompt comparisons of the SPM domain to the helix-loop-helix and leucine zipper domains used for homotypic and heterotypic protein interactions in other transcriptional regulators. In addition to in vitro studies, we show colocalization of the Scm and ph proteins at polytene chromosome sites in vivo. We discuss the possible roles of the SPM domain in the assembly or function of molecular complexes of PcG proteins.

A complex genetic locus, polyhomeotic, is required for segmental specification and epidermal development in D. melanogaster

Two mutagenic events are required to make null mutations of polyhomeotic (ph), which suggests that the locus is complex. Amorphic mutations (ph degrees) die in mid-embryogenesis and completely lack ventral thoracic and abdominal epidermal derivatives, whereas single-event mutations lead to transformations similar to those of known dominant gain of function mutants in the Antennapedia and bithorax complexes. After a chromosomal walk, the ph gene was localized using deficiencies and ph mutations that result from DNA rearrangements. Hybridization analyses show that there are two large, duplicated sequences in the ph region, and DNA lesions affecting either one of these repeats alter the function of the ph locus. We propose a model that may account for this unusual functional organization.

Maintenance of gene expression patterns.

In development, cells pass on established gene expression patterns to daughter cells over multiple rounds of cell division. The cellular memory of the gene expression state is termed maintenance, and the proteins required for this process are termed maintenance proteins. The best characterized are proteins of the Polycomb and trithorax Groups that are required for silencing and maintenance of activation of target loci, respectively. These proteins act through DNA elements termed maintenance elements. Here, we re‐examine the genetics and molecular biology of maintenance proteins. We discuss molecular models for the maintenance of activation and silencing, and the establishment of epigenetic marks, and suggest that maintenance proteins may play a role in propagating the mark through DNA synthesis. Developmental Dynamics 232:633–655, 2005.

The Drosophila polycomb group protein Psc contacts ph and Pc through specific conserved domains.

ABSTRACT The Polycomb group proteins are transcriptional repressors that are thought to act through multimeric nuclear complexes. We show that ph and Psc coprecipitate with Pc from nuclear extracts. We have analyzed the domains required for the association of Psc with ph and Pc by using the yeast two-hybrid system and an in vitro protein-binding assay. Psc and ph interact through regions of sequence conservation with mammalian homologs, i.e., the H1 domain of ph (amino acids 1297 to 1418) and the helix-turn-helix-containing region of Psc (amino acids 336 to 473). Psc contacts Pc primarily at the helix-turn-helix-containing region of Psc (amino acids 336 to 473), but also at the ring finger (amino acids 250 to 335). The Pc chromobox is not required for this interaction. We discuss the implication of these results for the nature of the complexes formed by Polycomb group proteins.

Site-Specific Recognition of a 70-Base-Pair Element Containing d(GA)n Repeats Mediates bithoraxoid Polycomb Group Response Element-Dependent Silencing

ABSTRACT Polycomb group proteins act through Polycomb group response elements (PREs) to maintain silencing at homeotic loci. The minimal 1.5-kb bithoraxoid (bxd) PRE contains a region required for pairing-sensitive repression and flanking regions required for maintenance of embryonic silencing. Little is known about the identity of specific sequences necessary for function of the flanking regions. Using gel mobility shift analysis, we identify DNA binding activities that interact specifically with a multipartite 70-bp fragment (MHS-70) downstream of the pairing-sensitive sequence. Deletion of MHS-70 in the context of a 5.1-kb bxd Polycomb group response element derepresses maintenance of silencing in embryos. A partially purified binding activity requires multiple, nonoverlapping d(GA)3repeats for MHS-70 binding in vitro. Mutation of d(GA)3repeats within MHS-70 in the context of the 5.1-kb bxd PRE destabilizes maintenance of silencing in a subset of cells in vivo but gives weaker derepression than deletion of MHS-70. These results suggest that d(GA)3 repeats are important for silencing but that other sequences within MHS-70 also contribute to silencing. Antibody supershift assays and Western analyses show that distinct isoforms of Polyhomeotic and two proteins that recognize d(GA)3 repeats, the TRL/GAGA factor and Pipsqueak (Psq), are present in the MHS-70 binding activity. Mutations inTrl and psq enhance homeotic phenotypes ofph, indicating that TRL/GAGA factor and Psq are enhancers of Polycomb which have sequence-specific DNA binding activity. These studies demonstrate that site-specific recognition of thebxd PRE by d(GA) n repeat binding activities mediates PcG-dependent silencing.