Epigenomic profiling of retinal progenitors reveals LHX2 is required for developmental regulation of open chromatin

Retinal neurogenesis occurs through partially overlapping temporal windows, driven by concerted actions of transcription factors which, in turn, may contribute to the establishment of divergent genetic programs in the developing retina by coordinating variations in chromatin landscapes. Here we comprehensively profile murine retinal progenitors by integrating next generation sequencing methods and interrogate changes in chromatin accessibility at embryonic and post-natal stages. An unbiased search for motifs in open chromatin regions identifies putative factors involved in the developmental progression of the epigenome in retinal progenitor cells. Among these factors, the transcription factor LHX2 exhibits a developmentally regulated cis-regulatory repertoire and stage-dependent motif instances. Using loss-of-function assays, we determine LHX2 coordinates variations in chromatin accessibility, by competition for nucleosome occupancy and secondary regulation of candidate pioneer factors.

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Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Auxiliary data, SRA files and tables have been deposited in the GEO repository, Accession Number GSE99818 at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE99818. All data needed to evaluate the conclusions in the paper are present in the paper, Supplementary Materials and Additional supplementary items.

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April 2018 All the figures and tables were derived from the data repository, except for Fig. S1 (flow sorting panels) and Supp.

Replication
See criteria for data inclusion/exclusion above Randomization n/a. See criteria for data inclusion/exclusion above.
Blinding N/A. Peak calling algorithms require to specify experimental condition and control samples so that normalization and enrichment of a given signal can be computed over stochastic distribution of the reads. Experimental conditions were always compared to control conditions for peak calling, differential accessibility of open chromatin regions or differentially expressed genes. IP fraction compared to isotype control, input samples (ChIP-Seq). GFP+ retinal progenitor cells fractions to GFP-post-mitotic fractions (RNA-Seq and ATAC-Seq).
Reporting for specific materials, systems and methods

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research Laboratory animals CD1 mice of either sex were euthanized at embryonic day 14 (E14) and postnatal day 2 (P2) according to Johns Hopkins IACUCapproved protocols. Timed pregnant CD-1 mice were obtained from Charles River Laboratories. Chx10-Cre:GFP mice were purchased from the Jackson Laboratories.

Wild animals
The study did not involve wild animals.

Field-collected samples
The study did not involve samples collected from the field.

ChIP-seq
Data deposition Confirm that both raw and final processed data have been deposited in a public database such as GEO.
Confirm that you have deposited or provided access to graph files (e.g. BED files) for the called peaks.

Sequencing depth
For each experimental condition involving a point-source factor and broad regions (ChIP-Seq and ATAC-Seq, respectively) a minimum of 20 Millions uniquely mappable reads or ≥10 millions uniquely mappable reads for each biological replicate were collected, according to the ENCODE's guidelines. For point-source datasets, non-redundant mapped reads were retained for downstream analysis. For ATAC-Seq, read length is 50bp, they are paired-end. For RNA-Seq, read length is 150bp, they are paired-end. For ChIP-Seq at E14, read length is 150bp, they are single-end. For ChIP-Seq at P2, read length is 100bp, they are single-end. For sequencing depth, percentage mappability and reproducibility please refer to Fig. S3D,E,F. Antibodies anti-Lhx2 c20 (Cat.# SC-19344X, TransCruz reagent, Santa Cruz Biotechnology): isotype control anti-goat (Cat.# ab37373, AbCAM); rabbit anti-H3K27Ac (Cat.# ab4729, Abcam); Rabbit IgG chip grade (Diagenode Cat #kch-504-250) Peak calling parameters Bowtie2 was used for ChIP-Seq reads alignment on the mouse genome (mm9). Uniquely mappable reads from ChIP-Seq were retained for peak calling by MACS2 (band width = 300, mfold =5, 50, d = 200, max tags per position =1, min FDR q-val cutoff = 1E-02, lambda = 1000-10000 bp). High-confidence ChIP-Seq peaks were identified from at least 2 experimental replicates (Poisson p-val threshold = 0.0001, min FE=4, FDR=0.001, max tags per position =1, normalization to input or isotype control Flow Cytometry Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation CD1 mice of either sex were euthanized at embryonic day 14 (E14) and postnatal day 2 (P2) according to Johns Hopkins IACUCapproved protocols. Timed pregnant CD-1 mice were obtained from Charles River Laboratories. Chx10-Cre:GFP mice (12) were purchased from the Jackson Laboratories. Retinas were freshly dissected, incubated in a suspension of papain and DNAse for 30 min at 37 C, inactivated with bovine serum albumin, resuspended in equilibrated Earle's balanced salt solution and subject to fluorescence activated cell sorting (98-99% purity) where viability was assessed by propidium iodide exclusion. Cell fractions were collected on poly-D-lysine coated slides, fixed in 4% paraformaldehyde for 10 min, permeabilized in TritonX-100 and stained for Chx10 (Cat.# X1179P, Exalpha), GFP (Cat.# 600-101-215, Rockland), Ki67 (Cat.# RM-9106-S1,Thermo Scientific) or Ccnd1 (Cat.# SC-450, Santa Cruz). The brightest fraction, differing of 4-fold mean intensity for GFP relative to the dim fraction, was always retained for subsequent processing and hereafter referred to as GFP-positive, RPC-enriched fraction. Lhx2 conditional embryonic knockouts were obtained by crossing Chx10-Cre:GFP with Lhx2lox/lox mice, and harvesting at E14 (30). Postnatal Lhx2 knockouts were generated by electroporation of pCAG-Cre-GFP construct into P0.5 wild type CD1 animals or Lhx2lox/lox animals. Retinas were harvested at P2, dissociated, and GFP-positive electroporated cells were isolated by FACS.
Overall electroporation efficiency was 2-3%. Gating strategy E14 and P2 Chx10-Cre:eGFP retinas were dissociated, gated by GFP mean intensity and the brightest flow-sorted fraction retained for immunostaining with Chx10 and GFP. Chx10-Cre:eGFP dissociated retinas were gated based on relative GFP+ intensity. Dissociated cells were first empirically gated based on clusters of GFP+ intensity emerging before the saturation of the signal at the inflection points of the histogram of events. The GFP-positive, GFP-negative and the interposed, dim fractions were collected. The brightest fraction had higher size and lower granularity (FSC Lin/ SSC Lin) compared to the dim and negative fractions. The presumptive RPC fraction differs for 4-fold GFP intensity from the dim fraction and represents 60% of the overall cell population. Cell viability was assessed by propidium iodide exclusion. Representative plots are from P2 retinas. The brightest GFP+ fraction was stained for Chx10, GFP and Ki67 or Ccnd1. The brightest fractions were hereafter referred to as GFP+, were always separated from the dim fraction and retained for subsequent applications. Control and Lhx2lox/lox retinas were electroporated at P0 with pCAG-Cre; GFP construct, collected after 48 hrs and subject to flow sorting. Viability was assessed by propidium iodide exclusion.

Instrument
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