Pax3 cooperates with Ldb1 to direct local chromosome architecture during myogenic lineage specification

Chromatin looping allows enhancer-bound regulatory factors to influence transcription. Large domains, referred to as topologically associated domains, participate in genome organization. However, the mechanisms underlining interactions within these domains, which control gene expression, are not fully understood. Here we report that activation of embryonic myogenesis is associated with establishment of long-range chromatin interactions centered on Pax3-bound loci. Using mass spectrometry and genomic studies, we identify the ubiquitously expressed LIM-domain binding protein 1 (Ldb1) as the mediator of looping interactions at a subset of Pax3 binding sites. Ldb1 is recruited to Pax3-bound elements independently of CTCF-Cohesin, and is necessary for efficient deposition of H3K4me1 at these sites and chromatin looping. When Ldb1 is deleted in Pax3-expressing cells in vivo, specification of migratory myogenic progenitors is severely impaired. These results highlight Ldb1 requirement for Pax3 myogenic activity and demonstrate how transcription factors can promote formation of sub-topologically associated domain interactions involved in lineage specification.

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Sample size
No sample size calculation was performed. Sample size was determined based on our own experience with the techniques used in this study. Based on the standard deviation of our experimental observations, we are confident the sample size chosen was sufficient to elicit differences between control and treated group.
Data exclusions No data were excluded Replication ChIP-seq and HiChIP experiments were replicated by using an independent differentiation experiment. In some cases, samples were collected on different days and processed at the same time. ChIP-seq for H3K4me1 using dTAD lines was performed once but selected loci were validated in independent biological replicates. Similarly, gene expression analyses were performed on independent samples. Number of replicates for each experiment is reported in the relative figure legend.

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All animals were handled in strict accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies, and all animal work was approved by the University of Minnesota Institutional Animal Care and Use Committee (protocol number 1702-34580A).
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ChIP-seq Data deposition
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May remain private before publication. Peak calling parameters Peak calling was performed using MACS with the following parameters: --bw 300 -p 1e-3. Similar results were obtained by performing peak calling using QESEQ with the following parameters: transcription factors/cofactors -s 100 -c 15 -p 0.01; histones marks -s 100 -c 20 -p 0.001 (replicate 1) -s 100 -c 15 -p 0.001 (replicate 2). To identify the list of high confidence PAX3 and LDB1 peaks we performed 3 independent ChIP-seq experiments and, using the MACS output and the bedtools intersect function, only common regions between 2 experiments were further considered. SMC1 and CTCF peaks were defined as the common peaks among 2 independent ChIP-seq datasets (using bedtools intersect). In addition, peak lists were filtered (intersect -v option) for sites overlapping to peaks detected in the uninduced control ChIP-seq and in the mouse ChIP-seq black-list.

Data quality
For all libraries, the average quality scores for the pass filter reads was ≥Q30. FASTQC was used to assess quality of all libraries and no libraries were discarded due to low quality. All duplicated reads were removed before downstream analyses. Peaks were called using 2 different peak calling algorithms (MACS and QESEQ), which provided similar results. Only high confidence peaks, identified as called in 2 biological replicates, were used for the analyses. The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
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Sample preparation
Single cells were collected using trypsin, washed with PBS and filtered using a 70micron mesh to remove cell-clumps. If required, cells were fixed with 1% formaldehyde or paraformaldehyde before staining. Cells were incubated with anti-Fc receptor and FBS before adding specific antibodies. After staining, cells were washed with PBS and then resuspended in 5%FBS/PBS supplemented with Propidium Iodide (PI). PI was not used for fixed cells.