Distal Enhancer – Gene Interactions at the Lmo2 locus in Mouse Erythroid cells

Distal regulatory elements (DREs) have been identified upstream of the hematopoietic regulator Lim domain only 2 (Lmo2) gene in the human and mouse genomes. In this thesis I have investigated how these elements regulate Lmo2 transcription in erythroid cells. My results show that strong chromatin-chromatin interactions exist between the DREs and the Lmo2 gene promoter in erythroid cells. These interactions are absent from kidney cells that do not express Lmo2. Within the distal chromatin interaction cluster encompassing three of the DREs increased DNase I sensitivity, presence of high levels of H3K4me1, and binding of multiple transcription factors, p300, cohesin (RAD21) and CTCF are observed. CTCF bound regions are located between the farthest DRE and the neighboring Caprin1 promoter suggesting that CTCF insulates Caprin1 from the DREs. Hence, my data suggests that these DREs function through a chromatin looping mechanism supported by cohesin associated with CTCF and transcription factor bound regions.MAS

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

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

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

<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

Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins

We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols

Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins

<div><p>We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that <i>in vivo</i> biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 <i>in vivo</i> biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce <i>in vivo</i> biotinylated Fabs in <i>E</i>. <i>coli</i>. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols.</p></div

Overview of targets and their success rates.

<p>Overview of the 265 antigen domains included in the study and what protein families they belong to. Family success rate was calculated as percentage of targets that were successful in selections and which also yielded at least one antibody that passed cell-based validation.</p><p>Overview of targets and their success rates.</p