Highly Restricted Localization of RNA Polymerase II within a Locus Control Region of a Tissue-Specific Chromatin Domain

Abstract

RNA polymerase II (Pol II) can associate with regulatory elements far from promoters. For the murine ␤-globin locus, Pol II binds the ␤-globin locus control region (LCR) far upstream of the ␤-globin promoters, independent of recruitment to and activation of the ␤major promoter. We describe here an analysis of where Pol II resides within the LCR, how it is recruited to the LCR, and the functional consequences of recruitment. High-resolution analysis of the distribution of Pol II revealed that Pol II binding within the LCR is restricted to the hypersensitive sites. Blocking elongation eliminated the synthesis of genic and extragenic transcripts and eliminated Pol II from the ␤major open reading frame. However, the elongation blockade did not redistribute Pol II at the hypersensitive sites, suggesting that Pol II is recruited to these sites. The distribution of Pol II did not strictly correlate with the distributions of histone acetylation and methylation. As Pol II associates with histone-modifying enzymes, Pol II tracking might be critical for establishing and maintaining broad histone modification patterns. However, blocking elongation did not disrupt the histone modification pattern of the ␤-globin locus, indicating that Pol II tracking is not required to maintain the pattern.Although well-defined basal transcription factors are required for transcription initiation, the assembly of these factors on promoters is highly regulated through transcription factors bound at promoters and at distal enhancers and locus control regions (LCRs) (7, 23). Transcription factors recruit basal factors through protein-protein interactions (47) and indirectly by recruiting chromatin-modifying coactivators, which increase promoter accessibility (5, 32, 60). While it is easy to envision how promoter-bound factors activate transcription, the mechanisms controlling transcription over a long distance on a chromosome are poorly understood. Evidence strongly supports a looping mechanism, in which chromatin structure juxtaposes distal regulatory elements with the gene, thereby allowing contact between factors bound at distant sites (6).Looping has been implicated in the transcriptional control of the murine ␤-globin genes (8, 56). The ␤-globin locus consists of four genes arrayed in the order in which they are expressed developmentally (Fig. 1A). High-level transcription of the ␤-globin genes requires upstream DNase I-hypersensitive sites (HS1 to HS4) (12, 59) referred to as the LCR (Fig. 1A) (4,10,11,17). As shown for erythroid cells from murine fetal liver, the LCR is positioned close to the active adult ␤-globin genes but not the inactive embryonic genes (8, 56), despite the fact that the embryonic genes are closer to the LCR linearly on the chromosome. However, determinants for establishing looping have not been defined, nor it is known how looping activates transcription, even though factors that bind the LCR and promoter in vivo are known (14,25,33,52). Looping also occurs within the HNF4␣ locus, and a tracking mechanism was propos...

The mediator complex functions as a coactivator for GATA-1 in erythropoiesis via subunit Med1/TRAP220

“…Furthermore, our data reveal recruitment of polII to an enhancer far away from the core promoter. Similarly, it has been demonstrated that polII is recruited to the ␤-globin locus control region, and that this occupancy depends at least in part on the presence of GATA-1 (38). Further support for polII occupancy at enhancers has been described for the androgen-specific gene PSA and for the TCR-␤ locus, both supported by in vivo footprinting and ChIP experiments (reviewed in ref.…”

Interleukin (IL)-4-independent Maintenance of Histone Modification of the IL-4 Gene Loci in Memory Th2 Cells

“…These are located in the region where the intergenic transcripts were perfectly preserved in memory Th2 cells (downstream of the CNS1 site), and thus this could account for the continuous generation of high level intergenic transcripts observed in the region. Similar highly restricted localization of Pol II within a locus control region was reported in the ␤-globin gene (53). As Pol II is known to associate with histone-modifying enzymes (26), Pol II localization within a locus control region may have also a specific role in histone modification, such as H3-K4 methylation and H3-K9/14 acetylation.…”

BRG1 requirement for long-range interaction of a locus control region with a downstream promoter

“…As the LCR is a site of intergenic transcription (47-50), LCR-associated Pol II might also generate functional transcripts and/or alter chromatin structure in a transcriptiondependent manner. However, blocking Pol II elongation has little to no effect on the ␤-globin locus histone modification pattern (50). GATA-1 increases Pol II occupancy at the LCR, although Pol II occupies the LCR in GATA-1-null cells (30).…”