Upstream Distal Regulatory Elements Contact the <i>Lmo2</i> Promoter in Mouse Erythroid Cells

<div><p>The Lim domain only 2 (<i>Lmo2</i>) gene encodes a transcriptional cofactor critical for the development of hematopoietic stem cells. Several distal regulatory elements have been identified upstream of the <i>Lmo2</i> gene in the human and mouse genomes that are capable of enhancing reporter gene expression in erythroid cells and may be responsible for the high level transcription of <i>Lmo2</i> in the erythroid lineage. In this study we investigate how these elements regulate transcription of <i>Lmo2</i> and whether or not they function cooperatively in the endogenous context. <b>C</b>hromosome conformation capture (3C) experiments show that chromatin-chromatin interactions exist between upstream regulatory elements and the <i>Lmo2</i> promoter in erythroid cells but that these interactions are absent from kidney where <i>Lmo2</i> is transcribed at twelve fold lower levels. Specifically, long range chromatin-chromatin interactions occur between the <i>Lmo2</i> proximal promoter and two broad regions, 3–31 and 66–105 kb upstream of <i>Lmo2</i>, which we term the proximal and distal control regions for <i>Lmo2</i> (pCR and dCR respectively). Each of these regions is bound by several transcription factors suggesting that multiple regulatory elements cooperate in regulating high level transcription of <i>Lmo2</i> in erythroid cells. Binding of CTCF and cohesin which support chromatin loops at other loci were also found within the dCR and at the <i>Lmo2</i> proximal promoter. Intergenic transcription occurs throughout the dCR in erythroid cells but not in kidney suggesting a role for these intergenic transcripts in regulating <i>Lmo2</i>, similar to the broad domain of intergenic transcription observed at the human β-globin locus control region. Our data supports a model in which the dCR functions through a chromatin looping mechanism to contact and enhance <i>Lmo2</i> transcription specifically in erythroid cells. Furthermore, these chromatin loops are supported by the cohesin complex recruited to both CTCF and transcription factor bound regions.</p></div

Distal regulatory elements upstream of <i>Lmo2</i> overlap transcription factor bound regions in erythroid cells.

<p>The mouse <i>Lmo2-Caprin1</i> region on chromosome 2 is depicted with chromosome coordinates shown at the top. Two <i>Lmo2</i> promoters are indicated by red boxes. Distal regulatory element (DRE) homology regions are indicated by black boxes joined by a line to delineate the human enhancer construct used in the generation of transgenic mice. Mouse ENCODE ChIP-Seq data for p300 and DNaseI hypersensitivity are shown below the DRE track. Coloured boxes represent peaks identified from transcription factor ChIP-Seq data for erythroid (MEL and GIE-ER4) cells. Overlapping transcription factor peaks were identified at the 75 and 12 DRE. These regions were also occupied by p300 and showed increased sensitivity to DNaseI. The entire locus was marked with histone H3 lysine 4 monomethylation (H3K4me1). Proximal promoter (pP), distal promoter (dP), murine erythroleukemia cells (MEL), Transcription factors (TF).</p

<i>Lmo2</i> primary transcripts are abundant in erythroid cells.

<p>Primary transcript levels in adult mouse anaemic spleen (red) and kidney (blue) for: <i>Lmo2</i> (exon2-intron2), <i>Caprin1</i> (exon3-intron2), <i>Slc4a1</i> (exon1-intron1), <i>Pkd2</i> (intron2-exon3), <i>Epn1</i>(exon1-intron1), <i>Gapdh</i> (exon1-intron1) and <i>Vh16 (genic)</i>. Levels were quantitatively assessed by RT-qPCR and expressed relative to <i>Gapdh</i>. <i>Epn1</i> is a second ubiquitously expressed reference gene, <i>Slc4a1</i> is an erythroid cell specific transcript, <i>Pkd2</i> is a kidney specific transcript, <i>Vh16</i> is not expressed in either tissue.</p

Intergenic transcription occurs upstream of <i>Lmo2</i> in erythroid cells.

<p>The mouse <i>Lmo2-Caprin1</i> region on chromosome 2 is depicted with chromosome coordinates shown at the top. RNA-Seq data for mouse fetal liver erythroblasts from Pilon et al. 2011 was obtained from the PSU Genome Browser (replicate 1 is shown in black). Transcript levels in adult mouse anaemic spleen and kidney were quantitatively assessed by RT-qPCR (shown in blue) and depicted relative to <i>Gapdh</i>. The levels downstream of 12 DRE (21.57) and the <i>Lmo2</i> pP (15.07) relative to <i>Gapdh</i> are off scale. Distal regulatory elements (DRE), distal promoter (dP), proximal promoter (pP).</p

CTCF and RAD21 are bound within the <i>Lmo2-Caprin1</i> region at sites of chromatin-chromatin interaction.

<p>The mouse <i>Lmo2-Caprin1</i> region on chromosome 2 is depicted with chromosome coordinates shown at the top. <i>Lmo2</i> promoters are indicated by red boxes. Distal regulatory element (DRE) homology regions are indicated by black boxes joined by a line to delineate the human enhancer construct used in the generation of transgenic mice. Mouse ENCODE ChIP-Seq data for the cohesin complex member RAD21 and CTCF are shown below DRE. Proximal promoter (pP), distal promoter (dP), murine erythroleukemia cells (MEL differentiated with 2% DMSO).</p

Distal regulatory elements interact with the <i>Lmo2</i> proximal promoter in erythroid cells.

<p><b>A</b>) Quantitative chromosome conformation capture (3C) was performed to detect chromatin-chromatin interactions between the 75 DRE (distal regulatory element) upstream of <i>Lmo2</i> and the rest of the <i>Lmo2</i>-<i>Caprin1</i> region of mouse chromosome 2. B) Similarly, 3C was performed to detect chromatin-chromatin interactions between the <i>Lmo2</i> proximal promoter (pP) and distal regulatory elements (DRE). In both the profile of interactions identified in anaemic spleen (red) and kidney (blue) is displayed. Black box indicates the anchor fragment at the 75 DRE or the <i>Lmo2</i> pP and alternating intensities of grey boxes indicate the fragments investigated for interactions. Data points are an average of three to five independent biological replicates. Error bars depict the SEM, * p<0.05, ** p<0.01, and *** p<0.001.</p

RNAPII is associated with enhancer regions.

<p>A) The <i>Hbb</i> (β-globin) LCR, located upstream of the <i>Hbb</i> genes, contains six characterized erythroid-specific DNase I hypersensitive sites (HS1-6). Peaks of RNAPII (green) identified using SISSRs overlapped HS1-4. Erythroid-expressed transcription factors have also been found associated with the LCR, overlapping the HS and RNAPII peaks. RNAPII ChIP sequences are shown in green, genomic DNA input sequences are shown in black and nucRNA sequences (only three in this region) are shown in blue. B) Distribution of RNAPII+/nucRNA- peaks relative to annotated genes. Roughly half of the RNAPII peaks identified by SISSRs are located in intergenic regions with 32.5% located more than 10 kb from an annotated gene (intergenic). C) Overlap of RNAPII+/nucRNA- peaks with erythroid-expressed transcription factors and conserved regions. D) An RNAPII+/nucRNA- peak 77 kb upstream of the <i>Lmo2</i> gene overlaps TF binding sites and is homologous to a validated enhancer identified in the human genome. Enhancer homology regions are indicated by black boxes joined by a line to delineate the human enhancer construct used in the generation of transgenic mice. NucRNA and RNAPII peaks surrounding the <i>Lmo2</i> gene are shown in blue and green respectively.</p

Outline of the experimental strategy.

<p>The nuclear transcriptome as well as RNAPII-associated genomic sequences of actively transcribing cells are analysed by nucRNA-Seq and RNAPII ChIP-Seq, respectively, as indicated. Top: schematic representation of transcription in the nucleus: four transcribing RNAPII complexes depicted as green shapes are associated with two chromatin fibres, DNA shown in red and blue, respectively; a third chromatin region, which is not being transcribed, is shown with DNA in black; histone complexes are yellow circles, nascent transcripts are shown as thin wavy lines, colours corresponding to chromatin. The nucRNA-Seq procedure is outlined on the left; purified nuclear RNA from the two transcribed regions is shown as wavy or straight lines colour-coded as above, DNA is depicted as thicker lines, random primers are black arrows, a putative genomic region with aligned Illumina paired-end (PE) tags signifies nucRNA-Seq data. The RNAPII ChIP-Seq procedure is outlined on the right; immunoprecipitated RNAPII-associated nucleosomes are depicted and colour-coded as above with cross-links as yellow crosses, anti-RNAPII antibodies are shown as red Y shapes, purified DNA is represented by thick lines, a putative genomic region with PE tags signifies RNAPII ChIP-Seq data.</p

Sequencing nuclear RNA reflects primary transcription at erythroid-expressed genes.

<p>A) Exonic vs intronic coverage for annotated genes in the 5′ (red), body (orange) and 3′ (yellow) regions by splitting each gene into equal thirds. B) Examples of RNA FISH signals for <i>Ank1</i> and <i>Gypa</i> shown in green, <i>Hbb-b1</i> is shown in red, nuclear DAPI staining is shown in blue, scale bar = 1 µm. C) Transcription frequency determined by RNA FISH compared to gene coverage in nucRNA-Seq data. We found a significant log-linear association between the transcription frequency determined by RNA FISH and the maximum nucRNA coverage depth (r_s = 0.820, 95% CI [0.582, 0.928], p<0.01).</p

Transcribed intergenic regions correspond to long non-coding RNAs.

<p>A) Nuclear vs cytoplasmic distribution for lncRNA candidates determined by RT-qPCR. B) Stability of nuclear retained lncRNA candidates was assessed by treatment with ActD for 1 and 4 hrs. Transcript levels were determined by RT-qPCR. Intranuclear distribution of lncRNA candidates was determined by RNA FISH for: C) lncRNA1 (Malat1), D) lncRNA2 (Neat1), E) lncRNA9, and F) lncRNA11, scale bar = 2 µm.</p