Bruce H. Howard, MD, Chief

The Laboratory of Molecular Growth Regulation (LMGR) conducts research on the control of cell proliferation, DNA replication, and gene regulation. With respect to the last, independent groups work in complementary areas, including regulation of the developing immune system, gene expression during early embryogenesis, chromatin-mediated gene silencing, and transcription of small RNA-encoding genes.

Keiko Ozato's group, the Section on Molecular Genetics of Immunity, investigates transcription factors that control cell proliferation and differentiation in the immune system. Studies on ICSBP/IRF-8 have revealed its importance for establishment of innate immunity through cytokine IL-12-dependent pathways. Photobleaching experiments showed that IRF-8 scans the genome by transiently binding to chromatin in macrophages and dendritic cells and demonstrated further that binding events are regulated by external cytokine signaling. These studies open a new way to study transcription in living immune cells. The group showed that Brd4, a second transcription factor under investigation, recognizes acetylated chromatin and remains bound to chromosomes during mitosis, implying a role in epigenetic memory. Brd4 recruits the key regulatory factor P-TEFb, a Cdk9/CyclinT heterodimer, which is essential for RNA polymerase II transcription elongation.

The investigations carried out by the Section on Eukaryotic Gene Regulation, led by Mel DePamphilis, focus on control of gene expression and DNA replication during early mouse embryogenesis, particularly the differentiation of trophoblast stem (TS) cells and embryonic stem (ES) cells. Of special interest is the endoreplication of the genome during the differentiation of trophoblast giant cells. A putative secreted signaling molecule, Dickkopf-like 1 (DkkL1), was characterized as a novel acrosomal protein expressed during sperm development. An independent project investigates cell cycle control and the role of the ORC complex in mammalian cell DNA replication. Orc1 is activated by ubiquitination and phosphorylation during the M- to G1-phase transition, whereas unmodified Orc1 can induce apoptosis. The BAH domain in human Orc1 promotes the association of ORC, more specifically the ORC(2-5) core complex, with chromatin; thus, this subunit appears to be directly involved in "origin recognition." These aspects of ORC1 regulation reflect its likely role as a link between DNA replication and mammalian development.

Another area of study relates to the multifunctional factor known as human La antigen. Work in the Section on Molecular and Cell Biology, under the direction of Richard Maraia, has shown that La antigen is a regulatory phosphoprotein that serves as a termination factor for RNA polymerase III, stimulating transcription and directing the post-transcriptional maturation of transcripts. A non-phosphorylated form of La is localized to the cytoplasm where it interacts with certain cellular and viral transcripts, including HIV mRNA. The group demonstrated that La is essential for development beyond the blastocyst stage of growth as well as for establishment of mouse embryonic stem cell lines. Further, the group mapped the pre-tRNA chaperone and RNA 3′ end protection activities of La to different motifs within the protein.

David Clark's Section on Chromatin and Gene Expression brings to the LMGR expertise in both yeast genetics and the characterization of purified native chromatin structures. High-resolution studies with the latter have revealed that gene activation can be accompanied by SWI/SNF- and Isw1-dependent chromatin remodeling over entire target gene domains. Remodeling involves large-scale movements of nucleosomes and conformational changes within nucleosomes. Related studies involve Spt10, a global regulator of core promoter activity. Intriguingly, Spt10 was found to bind with high specificity to the core histone promoter consensus motif (G/A)TTCCN6TTCNC. This work suggests that global changes in gene expression in Spt10-minus cells are indirect effects of defective regulation at the core histone genes.

A major effort of the Human Genetics Section, led by Bruce Howard, focuses on higher-order chromatin structure, particularly how defects in the maintenance of such structures (or failures in programmed transitions, especially in the perinatal period) may underlie common developmental disorders and age-related diseases. Random genome sampling, customized search algorithms for comparisons of annotated genomes, and genomics-style high-throughput approaches facilitate the detection and mapping of age-related areas of chromatin remodeling.