Milavetz Lab

Overview of Research Interests

Our lab is one of only a few investigating epigenetic regulation during viral infections and is arguably at the forefront of this research in the DNA tumor virus field. We use SV40 as a model to investigate the role of epigenetics in the regulation of eukaryotic replication and transcription. More recently we have begun to investigate the hold of epigenetics in the regulation of biological events which occur during the infectious cycle of the papovaviruses. We have shown that histone hyperacetylation in SV40 is dynamic and dependent upon the presence of p300 and a histone deacetylase co-migrating with the RNAPII transcription complex and proposed that a cell undergoing infection can recognize the incoming viral chromatin as foreign based upon its epigenetic marks and target the viral chromatin for inactivation or destruction. We routinely use many of the techniques which are utilized to study epigenetic regulation including many types of chromatin immunoprecipitation procedures, real time PCR, and siRNA inhibition.

Studies on Histone Hyperacetylation

Compaction of DNA by histone proteins helps to package the DNA into the nucleus at the expense of acting as a barrier to biological processes that require access to the DNA. Addition and removal of acetyl groups from the histone tails with the help of enzymes known as histone acetyltransferases (HATs) and histone deacetylases (HDACs) appear to play a critical role in maintaining the structure of chromosomes and the regulation of gene expression. Hyperacetylation of histones have been previously reported to cause an increase in the transcription of specific genes. In order to better understand the role of histone hyperacetylation in the regulation of gene expression we have been studying histone hyperacetylation in Simian Virus 40 (SV40) chromosomes during the course of a lytic infection using modified chromatin immunoprecipitation techniques. After determining the organization of hyperacetylated histones H4 and H3 and RNA Polymerase II (RNAPII) in unfractionated SV40 chromosomes using the ISF/CFIP technique we focused our attention to the role of histone hyperacetylation specifically during transcription (Ref ). Histone hyperacetylation in transcribing SV40 chromosomes was characterized utilizing a strategy in which the SV40 chromosomes undergoing transcription operationally defined by the presence of RNAPII were immune-selected with antibody to RNAPII and subjected to secondary chromatin immunoprecipitation with antibodies to hyperacetylated or unacetylated H4 or H3. Immune Selection Fragmentation and Immunoprecipitation (ISFIP) was used to determine the hyperacetylation status of histones independent of the location of the RNAPII and ReChromatin Immunoprecipitation (ReChIP) was used to determine their hyperacetylation status when associated with RNAPII. While hyperacetylated H4 and H3 were found in the coding regions regardless of the location of RNAPII, unacetylated H4 and H3 were only found at sites lacking RNAPII. The absence of unacetylated H4 and H3 at sites containing RNAPII was correlated with the specific association of the Histone Acetyl Transferase (HAT) p300 with the RNAPII. In contrast, the presence of unacetylated H4 and H3 at sites lacking RNAPII was shown to result from the action of a histone deacetylase (HDAC) based upon the effects of the inhibitor sodium butyrate. These results suggest that the extent of hyperacetylation of H4 and H3 during transcription alternates between hyperacetylation directed by an RNAPII associated HAT and deacetylation directed by an HDAC at other sites (Ref).We also studied dynamic histone hyperacetylation by using inhibitors of transcription such as alpha amanitin and DRB to block the translocation of RNAPII in the coding region. By blocking translocation of RNA Polymerase II using these inhibitors we have shown that there is a significant increase in the amount of unmodified H4 and H3 and in the case of inhibition by alpha amanitin also the loss of p300 from the RNAPII complex. In order to confirm that p300 plays a critical role in SV40 transcription, we have used siRNA to knockdown the expression of p300 and showed that there is little if any SV40 late transcription. We believe that these results demonstrate that histone hyperacetylation is dynamic in the coding region of genes with p300 causing the hyperacetylation and an as yet unknown HDAC responsible for the observed deacetylation (Ref)

Selected Publications

1. Balakrishnan L and Milavetz B (2010) (Invited Review, Critical Reviews in Biochemistry and Molecular Biology, Editor: Michael M Cox), Methylation of Histone H4, 45(5):440-52.
2. Balakrishnan L and Milavetz B (2010) Histone lysine 20 mono- and trimethylation define distinct biological processes in SV40 minichromosomes. Cell Cycle. 9(7). PubMed
3. Labyed Y, Kaabouch N, Schultz R, Singh B, and Milavetz B (2009) An improved 1-D gel electrophoresis image analysis system. Advantages in Computational Biology. Editor Springer. PubMed
4. Balakrishnan L and Milavetz B (2009) Dual Agarose Magnetic (DAM) ChIP. BMC Research Notes. 2(1):250. PubMed
5. Balakrishnan L and Milavetz B (2008) HDAC inhibitors stimulate viral transcription by multiple mechanisms. Virology Journal. 5:43. Virology Journal
6. Balakrishnan L and Milavetz B (2007) Histone Hyperacetylation during SV40 Transcription Is Regulated by p300 and RNA Polymerase II Translocation. Journal of Molecular Biology. 371(4), 1022-37. PubMed
7. Balakrishnan L and Milavetz B (2007) Histone Hyperacetylation in the coding region of chromatin undergoing transcription in SV40 minichromosomes is a dynamic process directly regulated by the presence of RNA Polymerase II. Journal of Molecular Biology. 365(1) 18-30. ScienceDirect
8. Balakrishnan L, Clauson R, Weiland T, Bianco M, and Milavetz B (2006) Sexually transmitted human papillomavirus type variations resulting in high grade cervical dysplasia in North-East North Dakota and North-West Minnesota. Virology Journal. 3:46. PubMed
9. Balakrishnan L and Milavetz B (2006) Reorganization of RNA polymerase II on the SV40 genome occurs coordinately with the early to late transcriptional switch. Virology. 345(1):31-43. PubMed
10. Balakrishnan L and Milavetz B (2005) Programmed Remodeling of Hyperacetylated Histone H3 and H4 Organization on the SV40 genome during lytic infection. Virology. 334 (1):111-123. PubMed