Genome Architecture Mediates Transcriptional Control of Human Myogenic Reprogramming

Abstract

Genome architecture has emerged as a critical element of transcriptional regulation, although its role in the control of cell identity is not well understood. Here we use transcription factor (TF)-mediated reprogramming to examine the interplay between genome architecture and transcriptional programs that transition cells into the myogenic identity. We recently developed new methods for evaluating the topological features of genome architecture based on network centrality. Through integrated analysis of these features of genome architecture and transcriptome dynamics during myogenic reprogramming of human fibroblasts we find that significant architectural reorganization precedes activation of a myogenic transcriptional program. This interplay sets the stage for a critical transition observed at several genomic scales reflecting definitive adoption of the myogenic phenotype. Subsequently, TFs within the myogenic transcriptional program participate in entrainment of biological rhythms. These findings reveal a role for topological features of genome architecture in the initiation of transcriptional programs during TF-mediated human cellular reprogramming.

Keywords: Integrative Aspects of Cell Biology; Molecular Structure; Omics; Systems Biology.

Graphical abstract

Figure 1

Myogenic Reprogramming of Human Fibroblasts (A) Time course of MYOD1-mediated reprogramming. The time window outlined in green corresponds to time points at which both genome architecture and transcription were captured by Hi-C (single replicates) and RNA-seq (in triplicate). (B) Scale-adaptive Hi-C matrices and gene expression. The considered scales include 1 Mb, 100 kb, TAD, and gene level.

Figure 2

Network Representation of Genomic Time Series Data (A) Mapping genomic form (Hi-C) and function (RNA-seq) to network architecture and node dynamics. Top left: Hi-C contact map (Toeplitz normalized, Methods) and RNA-seq at 100 kb resolution for chromosome 19. Top right: Network representation in which edge width indicates the Hi-C contact number and node color implies the magnitude of RNA-seq FPKM value. Bottom left: Network features given by eigenvector centrality, degree centrality, and betweenness centrality scores. The bars marked by different colors correspond to maximum centrality values. Bottom right: An illustrative network under different centrality measures. (B) Eigenvector centrality indicates chromatin compartments, termed A and B. Top left: Hi-C contact map of chromosome 3 at 100 kb resolution. Bottom left: RNA-seq, the first principal component (PC1) of the Hi-C correlation matrix, and eigenvector centrality (in terms of its Z score). Right: Correlation between RNA-seq, PC1, and eigenvector centrality extracted from Hi-C data for all chromosomes. Eigenvector centrality is a better indicator for chromatin compartments, marked by asterisk. (C) Betweenness centrality indicates A/B switched loci. Top left: Hi-C contact map of chromosome 19 (100 kb resolution) at time points 0 and 40 hr. Bottom left: A/B partition and betweenness centrality (in terms of its Z score) at 0 and 40 hr. The blue color represents A/B switched bins from 0 to 40 hr. The switched loci tend to have large betweenness centrality scores. Right: Significance of betweenness centrality at A/B switched loci. The p value is determined by comparing the average betweenness value at A/B switched bins with a random background distribution of other centrality values under the same number of bins. p Values are computed for all chromosomes and shown through an error bar plot in which the circle represents the p value averaged over all chromosomes and the horizontal error bar is determined by the SD of p values for all chromosomes.

Figure 3

Changes in Genome Architecture Precede Activation of the Myogenic Program (A) Genomic architecture (form) and gene expression (function) given by a Hi-C contact map and RNA-seq. Hi-C and RNA-seq are constructed at gene-level resolution. (B) Function and form change at successive time points evaluated by temporal difference score (TDS; Methods) of RNA-seq and network centrality features of Hi-C data, respectively. The significant form change (at 8 hr) occurs before the function change (at 16 hr). (C) Illustration of function TDS from 8 to 16 hr. Genes are divided into 2 clusters by applying K-means to their TDS values. Cluster 1 contains genes with the largest temporal change in RNA-seq. The gene expression can either decrease or increase from 8 to 16 hr. (D) Illustration of form TDS from 0 to 8 hr. Two gene clusters are obtained by applying K-means to their TDS values. Hi-C contact maps associated with a subset of genes in cluster 1 are shown from 0 to 8 hr, where the blue color indicates the Hi-C difference between the 2 time points. (E) Form-function change indicators for gene modules of interest during cellular reprogramming (top) and fibroblast proliferation (bottom), respectively. Here each row represents one gene module of interest, each column represents a time step, and the amount of change, as a percentage of total change over time for each module, is depicted by color. Percentage is determined by finding the number of genes with significant form-function change for each module and time step and dividing this number by the total number of significant gene changes for each module over time (row).

Figure 4

Fibroblasts Navigate a Critical Transition En Route to the Myogenic Lineage (A) Cell state trajectory of MYOD1-mediated reprogramming and fibroblast proliferation (Chen et al., 2015). Ellipsoids represent low-dimensional data representations obtained by applying Laplacian eigenmaps (Methods) to network form-function features. The branching trajectory shows a critical transition, or bifurcation, at 32 hr (p < 0.01). (B) Portrait of 4DN in the context of reprogramming and proliferation, respectively. It is described by a form-function domain (2D), constructed from 8 time points, for each chromosome. The fitted ellipsoid is obtained from the MVE estimate (Methods). (C) Shift of form-function domains of chromosomes at 32 hr. Chromosomes 5, 12, and 13 show the most significant changes of all chromosomes. (D) Form-function differences between cellular reprogramming and fibroblast proliferation, indicated by centroids and volumes of form-function ellipsoids for each chromosome. Top: Comparison between form change (horizontal shift) and function change (vertical shift) for each chromosome. Bottom: Variance of 4DN, given by volumes of chromosome ellipsoids under different cell dynamics.

Figure 5

Increased Genomic Contacts among Myogenic Regulatory Elements Set the Stage for Reprogramming (A) Early-phase expression dynamics of genes related to muscle cell terminal differentiation and chromatin remodeling. Genes encoding proteins involved in adult muscle function, including components of the contractile apparatus (DES, MYL4, TNNT1, TNN2), and EZH2, a repressor that is involved in myogenesis. (B) Chromatin remodeling factors and master transcription factors act cooperatively with MYOD1 to drive proliferating human fibroblasts into muscle cells. These factors include ARID5A, part of the BAF47 muscle remodeling complex that acts in cooperation with MYOD1; MEF2D, which drives differentiation of myotubes to skeletal and cardiac muscle; NR4A3 (aka NOR1) involved in differentiation of myotubes into smooth muscle; and SIX1, SIX4, and SOX4, which control the differentiation of myotubes into muscle cells. (C) Form and function of super enhancers and associated genes over time. Average Hi-C (read per million; RPM) contact between potential super enhancer and associated gene TSS regions over time, as defined by Hnisz et al. (2013). (D) Top upregulated SE-P genes, log₂(FPKM) (blue), and SE-P Hi-C normalized contact (red; see Methods) over time. (E) Four muscle-specific miRNAs have significantly increased expression levels in the later time points relative to the baseline control. X axis, sampling time points; y axis, log-scale differences at other time points compared with baseline (−48 hr).

Figure 6

Myogenic Genes Participate in Entrainment of Biological Rhythms (A) Gene network interactions between circadian E-box genes, derived from Ingenuity Pathway Analysis. (B) Core circadian gene expression over time. (B1) Dexamethasone synchronization. (B2) L-MYOD1 synchronization. Target and factor correspond to genes with E-box targets and TFs that bind to E-box genes, respectively. (C) Hi-C contacts between 26 core circadian genes over time (see Table S3). Rows and columns correspond to core circadian genes; contacts are binary (i.e., any contact between genes at a given time are shown). (D) Network connectivity of the largest connected component (LCC; Methods) of the studied Hi-C contact maps at different time points. (E) Normalized gene expression (FPKM, cubic spline) highlighting oscillation dampening after the bifurcation time 32 hr (red line) and the switch to differentiation medium for select core circadian genes; MYOD1 and MYOG also shown. (F) Normalized transcripts per million (TPM) of transcription factors that are targeted by MYOG or MYOD1 (ELF3) and that only showed oscillation after the critical transition at 32 hr (red line). (G) Conceptual diagram of biological rhythm entrainment during MYOD1-mediated reprogramming, where the red line signifies the bifurcation event.