Keiko Ozato, PhD, Head, Section on Molecular Genetics of Immunity
Tomohiko Tamura, MD, PhD, Staff Scientist
Anup Dey, PhD, Research Fellow
Akira Nishiyama, PhD, Research Fellow
Toru Atshumi, PhD, Visiting Fellow
Moon Kyoo Jang, PhD, Visiting Fellow
JiYoung Kim, PhD, Visiting Fellow
Hee Jeong Kong, PhD, Visiting Fellow
Hiroko Natsume, MD, PhD, Visiting Fellow
Prafullerkumur Tailor, PhD, Visiting Fellow
HongJie Yao, PhD, Visiting Fellow
Hormuzdir Dasenbrock, BS, Postbaccalaureate Fellow

We investigate transcription factors that regulate development of the immune system. IRF-8/ICSBP, a DNA-specific transcription factor, is required for the development of macrophages and dendritic cells (DCs). These cells express interferons (IFNs) and IL-12 cytokine genes and help establish innate immunity. To address the mechanism of IRF-8 action, we performed photobleaching experiments and showed that IRF-8 moves rapidly in the nucleus and scans the entire genome by transiently binding to chromatin in macrophages and DCs. Further analysis showed that external cytokine signaling regulates IRF-8-chromatin interactions. The results opened a new way to study transcription in living immune cells. We also investigate a model that deals with a bromodomain protein Brd4 that binds to acetylated chromatin and associates with chromosomes during mitosis. Brd4 is thought to play a role in epigenetic memory. We found that Brd4 interacts with P-TEFb, a Cdk9/CyclinT heterodimer that is essential for RNA polymerase II transcriptional elongation. Using chromatin immunoprecipitation (ChIP) analysis of the HIV-1LTR reporter, we provided evidence that Brd4 recruits P-TEFb to the promoter to stimulate transcription, pointing to a previously unknown role for Brd4 in transcription.

Real-time interaction of IRF-8 with chromatin in live macrophages

Laricchia-Robbio, ¹ Tamura, Kong, Tailor, Dasenbrock; in collaboration with Karpova, Levi, McNally, Xiong

Our recent microarray analysis showed that IRF-8 stimulates transcription of genes that are active in mature macrophages, including those encoding endosome/lysosome proteases. These genes are IRF8's immediate targets, given that IRF-8 stimulated them without resorting to intermediary factors. Interestingly, the genes' promoters contain a previously unknown IRF/Ets composite element. We showed that the composite element is the target of IRF-8 and that IRF-8 binds to the element by forming a complex with PU.1, a transcription factor of the Ets family. To address the mechanism by which IRF-8 regulates transcription of specific genes, we performed fluorescence recovery after photobleaching (FRAP). FRAP permits one to photobleach a small spot in the nucleus and then measure the recovery of fluorescence in the bleached spot. Previous studies have shown that many nuclear regulatory proteins are highly mobile and bind to chromatin in a transient manner; only constituents of chromatin such as core histones are shown to be immobile. Green fluorescent protein (GFP)-tagged IRF-8 (IRF-8/GFP) was expressed in IRF-8+/+ myeloid progenitor cells, and we monitored the mobility of IRF-8-GFP by FRAP as cells differentiated into macrophages. We found that IRF-8, like other nuclear proteins, was highly mobile and that the majority binds to chromatin very transiently, for less than 0.1 second. A small fraction of IRF-8 was less mobile and bound to chromatin for longer than 25 seconds at a time, although it also dissociated from chromatin in time. This dynamic chromatin binding property was a function of IRF-8 action in live macrophages and dendritic cells, given that (1) mutant IRF-8 without transcriptional activity was even more free-moving and exhibited no chromatin binding and (2) the mobility of IRF-8 was greater when tested in fibroblasts in which IRF-8 has no function. Furthermore, we showed that the mobility of IRF-8 was reduced when cells were stimulated by cytokine and pathogen signaling, indicating that IRF-8's chromatin-binding activity is directly linked to immunological stimuli. These observations provide a basis on which IRF-8 responds to signaling to regulate immune responses. We plan to extend photobleaching approaches to study the behavior of other IRF factors known to play a role in immune responses.

Interaction of Brd4 with P-TEFb: a role for Brd4 in RNA polymerase II-dependent transcription

Jang, Dey, Nishiyama, Yao; in collaboration with Brady, Yang, Zhou

While Brd4 interacts with acetylated chromatin, its precise role has remained elusive, although some studies implicated Brd4 in transcription and cell growth. In an effort to address Brd4's function, we searched for partner proteins that interact with Brd4. By immunopurification and mass-spectrometry analysis, we identified several candidate proteins that interact with Brd4. Among them, we found that P-TEFb is stably complexed with Brd4. P-TEFb is a Cdk9/cyclin T1 heterodimer that phosphorylates the CTD domain of RNA polymerase II and is critical for transcriptional elongation. We showed that bromodomains of Brd4 interact with cyclinT1 through the cyclin's small internal region outside the CDk9 binding region. It is known that P-TEFb complexes with the inhibitory subunit containing 7SK RNA and HEXIM1/2. Brd4 interacted with P-TEFb independently of the inhibitory subunit as a separate complex in the nucleus that was smaller than the inhibitory subunit-bound P-TEFb. Transfection experiments showed that Brd4 is required for P-TEFb's ability to stimulate transcription. We carried out the experiments with a reporter gene driven by the HIV-LTR promoter, the best-studied target of P-TEFb. We performed chromatin immunoprecipitation studies in cells in which Brd4 expression had been knocked down by shRNA. We found that P-TEFb is recruited to the HIV-1 LTR promoter through Brd4 and that Brd4-dependent P-TEFb recruitment is critical for transcription. These studies have established a role for Brd4 in RNA polymerase II-dependent transcription.

In a separate effort, we found that Brd4 plays a role in protecting cells from drug-induced mitotic stresses. To study the biological significance of Brd4-chromosome interaction during mitosis, we investigated the effect of anti-mitotic drugs, such as colchicine, nocodazole, and taxol, on Brd4-chromosome interactions and found that Brd4 is released from mitotic chromosomes when cells are treated with these drugs. Many other drugs that arrest mitotic processes similarly dissociate Brd4 from chromosomes. Dissociation of Brd4 is even more pronounced in cells in which expression of Brd4 is reduced as a result of disruption of a Brd4 allele (Brd4+/- cells). Furthermore, displacement of Brd4 from chromosomes coincided with increased apoptosis and reduced growth potential of drug-treated cells. Importantly, when cells with normal Brd4 expression were pretreated with nocodazole (a reversible anti-mitotic agent) and then incubated in fresh media, the large majority of cells proceeded through mitosis and produced new daughter cells. However, Brd4+/- cells with reduced Brd4 levels were defective in mitotic progression and produced fewer cells. The results indicate that the Brd4-chromosome interaction plays a protective role against drug-induced mitotic stress.

¹ Leopoldo Laricchia-Robbio, PhD, former Courtesy Contractor

COLLABORATORS

John Brady, PhD, Basic Research Laboratory, NCI, Bethesda, MD
Tatiana Karpova, PhD, Laboratory of Receptor Biology and Gene Expression, NCI, Bethesda, MD
Ben-Zion Levi, PhD, Technion-Israel Institute of Technology, Haifa, Israel
James McNally, PhD, Laboratory of Receptor Biology and Gene Expression, NCI, Bethesda, MD
Huabao Xiong, MD, PhD, City University of New York, Mount Sinai School of Medicine, New York, NY
Zhiyuan Yang, PhD, University of California, Berkeley, Berkeley, CA
Qiang Zhou, PhD, University of California, Berkeley, Berkeley, CA

For further information, contact ozatok@mail.nih.gov.