ONGOING RESEARCH AND FUTURE PLANS

1) Requirement for chromatin factors on target promoters during differentiation

The defining feature of differentiation process is activation of a cascade of genes.  There is quite poor understanding, however, how it is initiated and maintained.  We have discovered RBP2 is a part of gene-specific system of maintaining gene expression executed by trx genes. RBP2 ortholog in Drosophila, little imaginal discs (lid), since it belongs to a trx, a group of genes that has become a paradigm of transcriptional regulators on the level of chromatin.  Drosophila female sterile homeotic (fsh) locus interacts synergistically with trx loci (Gans et al.1980). Since its human homolog is a direct RBP2 target gene, RBP2 function might be related to the trx function.  This study has initiated a DNA-binding analysis that would be aimed to identify important gene mechanisms in differentiation in vivo.  For further characterization of the complexity of gene regulation by RBP2, we will look for various proteins recruited to the RBP2 target genes by ChIP analysis.  In addition, by analyzing histone modifications on RBP2 target genes, we hope to address the nature of the heritable mark(s) in differentiation.

We showed that pRB remains bound to chromatin during differentiation.  How pRB regulates its targets in differentiation is unknown.  This project seeks to determine these mechanisms.  For the first time it provides a way to identify non-E2F target genes regulated by pRB, which are important in vivo and under physiological conditions.  The results of my ChIP-on-chip analyses, combined with data on target gene functions available from public resources, will help to determine “decision points” in differentiation solved by RBP2.  I hypothesize that activation of some target genes by pRB in RBP2-dependent manner generates a persistent signal for differentiation.

2)  The mechanism of mediating differentiation by RBP2 

A number of my experiments attributed both repressing and activating activities to the RBP2.  First, we found that RBP2 has an opposing action on pRB-mediated activation of some transcription factors (Figure 4).  Second, we discovered another class of RBP2 target genes, typified by BRD2 and BRD8, where pRB and RBP2 cooperate to activate transcription rather than antagonizing one another.  How these activities can be attributed to the same protein and what is their biological significance is unknown.  My future study will, first, address the major question that stands out from my previous research of how RBP2 accomodates the two functions on different genes and what is the contribution of pRB to this ability.   I will be using immunoprecipitations experiments and ChIP analyses to investigate factors modifying these activities and the required developmental timing.  A fundamental problem is why some promoters appear to require different chromatin remodeling or modifying activities than the others.  All together, these data will provide some mechanistical clues on how different RBP2 complexes are involved in regulation of RBP2 target genes. 

3)  Functional overlap and differences between RBP2 family members

RBP2 has single homologs in other organisms.  In corn fungus, C. elegans and fly, RBP2 homologs have been described as developmental regulators.  In human, RBP2 has very high identity to three proteins: PLU-1, a gene that escapes X-chromosome inactivation Xe169, and its homolog on Y-chromosome ScmY.  Moreover, we described two RBP2 isoforms, with two and three PHD fingers, respectively.  The third PHD finger has recently been shown to be involved in translocation in a patient with acute myeloid leukemia (AML).  Given the significance of RBP2, what are the functions of these uncharacterized proteins?  How do they contribute to cancer development in different tissues?  The similarity between RBP2 family members points to a potential central mechanism of the involvement in chromatin-mediated transcriptional regulation.  For example, PLU-1 and RBP2 have transcriptional repression properties suggesting that they both recruit co-repressor complexes.  PLU-1 is a cancer antigen overexpressed in 90% of breast cancers.  Little is known about the biology of PLU-1 and whether it plays a causal role in breast epithelial transformation.  Based on similarity of PLU-1 to RBP2, I hypothesize that PLU-1, like RBP2, stimulates cell proliferation and impairs differentiation.  Moreover, I hypothesize that the elevated levels of PLU-1 observed in breast cancer cells might, like loss of pRB activity, contribute to breast epithelial transformation.

4) Proteins that mediate the ability of pRB to induce differentiation

I performed a screening of cDNA libraries using a modified yeast two-hybrid system with the pRB variant D663 that activates transcription/promotes differentiation but fails to repress E2F.  As a result, I isolated several clones, with the strongest positive encoding RBP2.  Plzf is known as a gene involved in rare translocations (t11,17) associated with the development of acute promyelocytic leukemia.  Plzf associates with repressor complexes and acts as a main upstream regulator of expression of Hox genes involved in limb and body patterning.  I have mapped the PLZF pRB-binding domain, and this suggests a strategy of disrupting the binding between PLZF and pRB in cells.  This will allow investigation into importance of PLZF to the biology of leukemia.  I think comprehensive analysis of all three positives is important, because they may uncover different aspects of pRB regulation.

5)  Another transcriptional function of the nucleolus 

The nucleolus is the most striking example of specialized transcription known as a fabric of ribosomal DNA transcription.  However, targeting to the nucleoli has been also described for a number of chromatin activities, including pRB, during acquisition competence for cell fate switch.  RBP2 is a protein of nucleolus in both undifferentiated and differentiated cells.  I would like to use a newly developed technique of nucleoli purification to investigate nucleoli function.  One of specific aims would be to study the pRB protein complexes as a function of differentiation using mass spectrometry analysis.