amm104

Alexander M. Mazo, PhD

Contact Dr. Mazo

1020 Locust Street
JAH, Rm. 485
Philadelphia, PA 19107

(215) 503-4785
(215) 923-7144 fax

Expertise and Research Interests

Transcriptional Regulation by Epigenetic Factors and Nuclear Hormone Receptors

Our primary research interests center on understanding of the transcriptional mechanisms during development of the eukaryotic organisms. Tight chromatin structure prevents transcription. Recent advances in this field have revealed that there are two major mechanisms that are employed by the cell to alleviate the repressive effects of chromatin, i.e. nucleosome remodeling by the ATP-dependent chromatin remodeling complexes and multiple types of modifications of nucleosomal histones. Modifications at histone tails by specific enzymes may lead either to repression or to activation of transcription. Most types of histone modifications are reversible, for example acetylation, phosphorylation and ubiquitination of histone tails, while methylation is an irreversible histone modification. It is thought that methylation of histone tails is especially important since it may irreversibly modify nucleosomes, thus creating an epigenetic mark that will be maintained during development, to lock-in a specific state of gene expression. Methylation of lysine residues in histone tails is achieved by the proteins containing a well-conserved SET domain. We have cloned and characterized a number of SET domain-containing proteins, including Trithorax (Trx), the first known protein of this kind.

Trx belongs to the Trithorax group (trxG) of chromosomal transcriptional regulators, which together with the genetically defined Polycomb group (PcG) of proteins are required for positive (trx-G) and negative (Pc-G) maintenance of expression of numerous developmental genes, including genes of the homeotic (HOX) complexes in Drosophila. Most of the members of both groups have homologues in higher organisms. For example, the MLL/ALL1/HRX protein is a structural and functional homologue of Trx, and it is also involved in regulation of HOX genes in mammals. The cytological region of the MLL gene is translocated in the majority of childhood leukemias, leading to a number of abnormal MLL fusion proteins, which are believed to play a major role in the disease. To understand the role of Trx in development, we purified the Trx protein complex. This protein complex, TAC1, possesses dual histone modification activities, methylation of lysine 4 of histone H3 via the activity of the SET domain of Trx and histone acetylation by dCBP that is associated in the same protein complex. Furthermore, both these activities are found to be important in regulating the Trx target genes. To better understand the way these activities are utilized during development, we determined the binding sites/response elements (TREs) for a number of the trxG proteins, including proteins of the Trx complex TAC1 in the homeotic gene Ultrabithorax (Ubx). The Trx-specific TREs were found in 25 kb upstream of the Ubx start site, and these regulatory elements are juxtaposed to other trxG and PcG (PRE) response elements. These findings fit well into our data that suggest that the Trx protein directly interacts with a number of other trxG and PcG proteins, the binding sites for which are found in the close vicinity to the Trx TRE. The challenge now is to understand what is the chromatin structure of the trxG/PcG target gene, when this gene is activated or silenced. This issue is relevant with respect to modifications of histone residues in the vitally important regulatory elements and with respect to the occupancy of these regulatory elements by the members of the two functionally opposite protein families, trxG and PcG. To get an insight into this issue we developed a cell-sorting procedure which allows to obtain the Ubx-expressing cells from live embryos, in amounts sufficient for biochemical analysis.

Recently, we have found that TAC1 protein complex is required to sustain high levels of heat shock gene expression following heat induction. This is an important finding since heat shock proteins are molecular chaperons that are central for many cellular processes, including a number of diseases. Besides, regulation of heat shock gene expression is one of the best-developed transcriptional systems, which would allow us to address the fundamental questions of Trx/TAC1 functioning. Our results suggest that TAC1 is recruited as a whole complex to a number of heat shock genes. In particular, it is recruited to the 5-coding region of hsp70. This recruitment induces TAC1-specific methylation and acetylation of histones in the same 5-coding region of hsp70. These data suggest that TAC1 complex may be involved in facilitating transcriptional elongation by RNA polymerase II (Pol II). A number of elongation factors, Spts and FACT, as well as the cyclinT-dependent Cdk9 kinase (P-TEFb complex), are also shown to facilitate elongation by Pol II. We are now interested in finding a potential link between the recruitment of TAC1 and other elongation factors, as well as a link between the activities of these elongation factors and the histone modifying activities of the TAC1 complex.

We have cloned two other SET domain-containing genes, the Drosophila Trithorax-related (TRR) and its mammalian homologue ALL1-related (ALR). We found that Drosophila TRR is also a specific lysine-4 histone H3 methyltransferase (HMTase), and it is a component of several high molecular weight complexes. TRR directly interacts with EcR and Usp, two components of the ecdysone hormone nuclear receptor complex. In larvae, these proteins are involved in the ecdysone-dependent development of the Drosophila eye through direct regulation of the major eye morphogen Hedgehog (Hh). This is the first example of the lysine HMTase, which acts as a direct co-activator of the hormone receptor. Consistent with this, activation of Hh expression is accompanied by the ecdysone- and TRR-dependent methylation of lysine-4 of histone H3 at the Hh promoter. Our genetic analyses of TRR mutants indicates that besides its role in regulation of Hh during eye development, TRR may be a component of another ecdysone-dependent, apoptotic pathway, which also includes p53. Clonal analysis of the TRR mutants also suggests that TRR and EcR may be involved in formation of the appendages in the Drosophila larval imaginal discs. While pursuing the genetic analysis of the TRR/EcR pathways, we are also engaged in the biochemical purification of the TRR protein complex. This work, as in case of Trx, may reveal additional TRR-related biochemical activities, via its association with other chromatin-modifying proteins. Indeed, our preliminary data indicates that TRR may be associated with another protein with specific histone acetylation activity. The role of the components of the purified TRR complex in development, the finding of other in vivo TRR target genes and EcR/TRR response elements remains the major focus of this project.

Keywords

epigentics; chromosomal proteins; homeotic genes; trithorax, Polycomb; nuclear hormone receptors; chromatin modifications