Mark Groudine

We are investigating the relationships among chromatin structure, transcriptional activators and the regulation of gene expression during erythroid differentiation. Using the human and mouse beta-globin loci as models, we have: (1) analyzed the function of locus control region (LCR) by targeted deletion in cell lines and mice (accomplished via homologous and site specific recombination); (2) determined the composition of erythroid specific transcription complexes prior to and after erythroid differentiation (by mass spectrometry); and (3) determined the binding of such complexes to the beta-globin locus regulatory elements by chromatin immune-precipitation (ChIP). We have combined these molecular and biochemical studies with fluorescence in situ hybridization (FISH) to visualize the nuclear location of the beta-globin loci and immunofluorescence studies to detect the subcellular localization of regulatory proteins during erythroid differentiation in vivo and in vitro. Our results suggest a multi-step model for gene activation, involving alterations in the nuclear location of the loci and transactivators, the binding of these factors to elements in the beta-globin loci, and the subsequent high level transcription of the beta-globin genes. Our work has also revealed that the mammalian interphase nucleus is organized in a non-random, tissue-specific fashion reflecting the genomic organization of genes that are co-regulated during differentiation.

Our principal findings include:

1. The LCR is essential for high level transcription of the beta-globin genes in differentiated erythroid cells. To determine the molecular mechanisms underlying LCR function, we investigated factor recruitment to the adult beta-globin gene in wild-type WT and locus control region knockout (DLCR) loci in mice. We found that while the LCR deletion has little effect on activator recruitment and pre-initiation complex (PIC) assembly, it has a dramatic effect on ser-5 phosphorylation of RNA polymerase II and transcriptional elongation. Thus in contrast to prevailing views, the LCR functions primarily downstream of activator recruitment and PIC assembly.

2. Looping of gene loci from their chromosome territories (CTs) in the interphase nucleus reflects poised and repressed states, in addition to the transcriptionally active state. We have found that wild type murine and human beta-globin loci are looped away from their CTs at a high frequency in an erythroblast cell background prior to activation of globin gene expression. Conversely, a mutant allele lacking the LCR (DLCR) does not loop from the CT. This result suggests that the LCR-dependent poising of the globin locus away from the CT may be essential for access to subsets of the transcriptional machinery prior to activated transcription. Replacement of the LCR with a B-cell specific regulatory element (IgH LCR) that represses gene activity in non-B cells also resulted in looping of the globin locus away from its CT into the repressive centromeric heterochromatin compartment. These results indicate that chromosome territory looping may play a significant role in cell-type specific transcriptional activation or repression of a locus.

3. Dynamic changes in transcription factor complexes accompany erythroid differentiation: Prior to erythroid differentiation, a heterodimer composed of the small Maf protein MafK and the repressor Bach1 recruits transcriptional co-repressor complexes to the beta-globin locus, resulting in repression of globin gene expression. Upon induction of erythroid differentiation, an exchange of MafK-binding partners occurs: Bach 1 is replaced by the transcriptional activator p45. This, in turn, leads to displacement of co-repressor complexes from the locus and recruitment of co-activators, resulting in globin gene expression. The mechanism behind this exchange involves the relocation of MafK in the nucleus: Prior to induction of differentiation, MafK co-localizes with centromeric heterochromatin, whereas p45 is restricted to euchromatic nuclear compartments. Terminal differentiation is accompanied by the relocation of MafK (and the beta-globin locus) to euchromatic regions and formation of the MafK/p45 heterodimer.

4. We observed that genes that are co-regulated during differentiation have a significant tendency to be proximally distributed along chromosomes. In turn, we found that the frequency at which homologous chromosomes associate is related to the number of co-regulated genes they possess. We therefore suggested that coordinate gene regulation during cellular differentiation may yield lineage-specific nuclear topologies that facilitate gene co-regulation. Moreover, we hypothesized that the process of self-organization is responsible for the emergence of these topologies. We have directly tested our idea that chromosomes self-organize during differentiation according to coordinate gene regulation by determining the collective similarity between gene regulatory and chromosomal association networks by expressing them as matrices. To construct the matrices, we measure the relative entropy--or ``distance''--among nodes within networks as well as between networks during differentiation, allowing us to assay shared global properties and the emergence of lineage-specific relationships. Our analysis has demonstrated that the networks of co-regulated gene expression and chromosomal association are indeed mutually related during differentiation, resulting in the self-organization of lineage-specific chromosomal topologies. Currently, we are testing our hypothesis that transcription factor complexes "link" co-regulated genes (on the same or different chromosomes), resulting in a tissue-specific nuclear organization that resembles a scale-free network.