Cohesin mediates chromatin interactions that regulate mammalian β-globin expression

Abstract

The β-globin locus undergoes dynamic chromatin interaction changes in differentiating erythroid cells that are thought to be important for proper globin gene expression. However, the underlying mechanisms are unclear. The CCCTC-binding factor, CTCF, binds to the insulator elements at the 5' and 3' boundaries of the locus, but these sites were shown to be dispensable for globin gene activation. We found that, upon induction of differentiation, cohesin and the cohesin loading factor Nipped-B-like (Nipbl) bind to the locus control region (LCR) at the CTCF insulator and distal enhancer regions as well as at the specific target globin gene that undergoes activation upon differentiation. Nipbl-dependent cohesin binding is critical for long-range chromatin interactions, both between the CTCF insulator elements and between the LCR distal enhancer and the target gene. We show that the latter interaction is important for globin gene expression in vivo and in vitro. Furthermore, the results indicate that such cohesin-mediated chromatin interactions associated with gene regulation are sensitive to the partial reduction of Nipbl caused by heterozygous mutation. This provides the first direct evidence that Nipbl haploinsufficiency affects cohesin-mediated chromatin interactions and gene expression. Our results reveal that dynamic Nipbl/cohesin binding is critical for developmental chromatin organization and the gene activation function of the LCR in mammalian cells.

FIGURE 1.

Adult β-globin gene induction in DMSO-treated MEL cells. A, schematic diagram of the mouse β-globin locus. The 80-kb β-globin locus is flanked by olfactory receptors and contains an upstream LCR and a globin gene cluster 12–50 kb downstream of the LCR. Several HS are located in the LCR. Most notably, HS2 functions as an enhancer, and the outer HS5 functions as an insulator. B, adult β-globin gene induction following DMSO treatment. End point RT-PCR of β-major gene induction and q-RT-PCR analysis of β-major and β-minor genes in comparison to GAPDH were examined over 4 days of DMSO treatment. Q-RT-PCR values were normalized to ribonuclease/angiogenin inhibitor 1 (rnh1). C, ChIP analysis of cohesin binding to the β-major promoter following DMSO induction from 0 to 4 days. End point PCR and semi-quantitation (Quantity One, Bio-Rad) of anti-Rad21 ChIP DNA are shown. Preimmune IgG ChIP was used as the negative control. Western blot analysis showed that the level of total Rad21 remains constant. α-tubulin was used as a loading control. D, ChIP analysis of cohesin (Rad21), Nipbl, CTCF, and NF-E2 binding to the β-globin locus. The locations of the primers are indicated at the bottom. HS5, CTCF insulator; HS2, enhancer; itg, intergenic region downstream of ϵy (see Fig. 1A and supplemental Fig. S2). NIH-3T3 fibroblast cells were used as a negative control. Arbitrary values were given for the y axis ((IP signal-Preimmune)/Input). E, 3C and ChIP-loop analysis of the HS2 and β-major promoter interaction at 4 days after DMSO treatment. The interactions of a region containing HS4 and HS5 (HS4/5) with either 3′HS1 or HS-62 are compared. An uncut region without 3C digestion sites on the odd-skipped-related 2 (osr2) promoter was used as loading control. For ChIP-loop analysis using anti-Rad21 antibody, the HS2-β-major interaction was specifically detected after DMSO induction. The HS2-ϵy interaction was examined for comparison. Preimmune IgG was used as the negative control for IP. The osr2 promoter is used again as loading control, as cohesin constantly binds to the osr2 promoter in both untreated and DMSO-induced MEL.

FIGURE 2.

Analysis of the effect of Nipbl/cohesin binding to the β-globin locus in vivo. A, ChIP analysis of RNA polymerase II, cohesin (Rad21), and CTCF at the DNase l hypersensitive sites in the β-globin region of E15.5 liver tissues. The PCR-amplified regions are indicated at the bottom (see also Fig. 1A). Q-PCR analysis of ChIP samples were normalized as in Fig. 1C. B, ChIP analysis of E15.5 liver using antibodies specific for Rad21, Nipbl, CTCF, and NF-E2. Q-PCR analysis of ChIP samples was normalized as in Fig. 1C. The age-matched Nipbl mutant liver and the wild-type brain were used for comparison. Similar results were obtained at E13.5. C, 3C analysis of the β-globin locus in E13.5 liver. Two different baits (HS2 and HS4/5) were used for the analysis. The results were compared with the age-matched liver tissue from the Nipbl mutant mice as indicated. D, Q-RT-PCR of the β-globin and Nipbl expression analysis of the wild-type and Nipbl mutant fetal liver at E11.5, E13.5, and E15.5. GAPDH was used for comparison. All genes were normalized to rnh1. E, ChIP analysis of RNA polymerase II (pol II) at the β-globin locus in the wild-type and Nipbl mutant E13.5 liver.

FIGURE 3.

Differential effect of CTCF and cohesin in chromatin interactions in human erythroid cells. A, ChIP analysis of cohesin and CTCF at the β-globin locus in 293T and K562 cells. Locations in the β-globin locus are indicated at the bottom. B, the effect of CTCF depletion on cohesin binding at the β-globin locus. Fold-change of cohesin binding at each site was compared between control siRNA and CTCF siRNA depletion. CTCF ChIP at its binding sites (HS5 and 3′HS1) was also examined for siRNA specificity. Western blot analysis of CTCF depletion is shown in supplemental Fig. S4A. C, 3C analysis of the effect of hSMC1 or CTCF siRNA depletion at the β-globin locus compared with control siRNA. Left, HS2/3 as bait; right, HS5 as bait. D, Q-RT-PCR analysis of embryonic (ϵ), fetal (Gγ), and adult (β) β-globin gene expression in 293T, K562, and K562 treated with control, hSMC1, or CTCF siRNA (see also supplemental Fig. S4). Expression was normalized against β-actin.

FIGURE 4.

A schematic diagram of cohesin function at the β-globin locus. In the silent β-globin locus of erythroid progenitors, cohesin and CTCF bind at the HS5 and 3′HS1 insulator sites, setting the boundaries for later activation. When the locus becomes active, more transcription factors are recruited, and they can strengthen the chromatin interactions for optimal gene expression. Cohesin has a dual function, as it mediates both gene-enhancer interactions and boundary insulator interactions.