Rewirable gene regulatory networks in the preimplantation embryonic development of three mammalian species.

Mammalian preimplantation embryonic development (PED) is thought to be governed by highly conserved processes. While it had been suggested that some plasticity of conserved signaling networks exists among different mammalian species, it was not known to what extent modulation of the genomes and the regulatory proteins could "rewire" the gene regulatory networks (GRN) that control PED. We therefore generated global transcriptional profiles from three mammalian species (human, mouse, and bovine) at representative stages of PED, including: zygote, two-cell, four-cell, eight-cell, 16-cell, morula and blastocyst. Coexpression network analysis suggested that 40.2% orthologous gene triplets exhibited different expression patterns among these species. Combining the expression data with genomic sequences and the ChIP-seq data of 16 transcription regulators, we observed two classes of genomic changes that contributed to interspecies expression difference, including single nucleotide mutations leading to turnover of transcription factor binding sites, and insertion of cis-regulatory modules (CRMs) by transposons. About 10% of transposons are estimated to carry CRMs, which may drive species-specific gene expression. The two classes of genomic changes act in concert to drive mouse-specific expression of MTF2, which links POU5F1/NANOG to NOTCH signaling. We reconstructed the transition of the GRN structures as a function of time during PED. A comparison of the GRN transition processes among the three species suggested that in the bovine system, POU5F1's interacting partner SOX2 may be replaced by HMGB1 (a TF sharing the same DNA binding domain with SOX2), resulting in rewiring of GRN by a trans change.

Dissecting early differentially expressed genes in a mixture of differentiating embryonic stem cells.

The differentiation of embryonic stem cells is initiated by a gradual loss of pluripotency-associated transcripts and induction of differentiation genes. Accordingly, the detection of differentially expressed genes at the early stages of differentiation could assist the identification of the causal genes that either promote or inhibit differentiation. The previous methods of identifying differentially expressed genes by comparing different cell types would inevitably include a large portion of genes that respond to, rather than regulate, the differentiation process. We demonstrate through the use of biological replicates and a novel statistical approach that the gene expression data obtained without prior separation of cell types are informative for detecting differentially expressed genes at the early stages of differentiation. Applying the proposed method to analyze the differentiation of murine embryonic stem cells, we identified and then experimentally verified Smarcad1 as a novel regulator of pluripotency and self-renewal. We formalized this statistical approach as a statistical test that is generally applicable to analyze other differentiation processes.

A Chemically Well-Defined, Self-Assembling 3D Substrate for Long-Term Culture of Human Pluripotent Stem Cells.

Clinical applications of human pluripotent stem cells (PSCs) are limited by the lack of chemically well-defined scaffolds for cell expansion, differentiation, and implantation. In this study, we systematically screened various self-assembling hexapeptides to identify the best matrix for long-term 3D PSC culture. Lysine-containing Ac-ILVAGK-NH2 hydrogels maintained best the pluripotency of human embryonic and induced PSCs even after 30 passages. This peptide matrix is also compatible with the use of xeno-free and defined differentiation media. By exploiting its stimuli-responsive sol-gel transition, arrays of encapsulated PSCs can be bioprinted for large-scale cell expansion and derivation of miniaturized organoid cultures for high-throughput screening.

Cross-species de novo identification of cis-regulatory modules with GibbsModule: application to gene regulation in embryonic stem cells.

We introduce the GibbsModule algorithm for de novo detection of cis-regulatory motifs and modules in eukaryote genomes. GibbsModule models the coexpressed genes within one species as sharing a core cis-regulatory motif and each homologous gene group as sharing a homologous cis-regulatory module (CRM), characterized by a similar composition of motifs. Without using a predetermined alignment result, GibbsModule iteratively updates the core motif shared by coexpressed genes and traces the homologous CRMs that contain the core motif. GibbsModule achieved substantial improvements in both precision and recall as compared with peer algorithms on a number of synthetic and real data sets. Applying GibbsModule to analyze the binding regions of the Krüppel-like factor (KLF) transcription factor in embryonic stem cells (ESCs), we discovered a motif that differs from a previously published KLF motif identified by a SELEX experiment, but the new motif is consistent with mutagenesis analysis. The SOX2 motif was found to be a collaborating motif to the KLF motif in ESCs. We used quantitative chromatin immunoprecipitation (ChIP) analysis to test whether GibbsModule could distinguish functional and nonfunctional binding sites. All seven tested binding sites in GibbsModule-predicted CRMs had higher ChIP signals as compared with the other seven tested binding sites located outside of predicted CRMs. GibbsModule is available at (http://biocomp.bioen.uiuc.edu/GibbsModule).

Unbiased mapping of transcription factor binding sites along human chromosomes 21 and 22 points to widespread regulation of noncoding RNAs.

Using high-density oligonucleotide arrays representing essentially all nonrepetitive sequences on human chromosomes 21 and 22, we map the binding sites in vivo for three DNA binding transcription factors, Sp1, cMyc, and p53, in an unbiased manner. This mapping reveals an unexpectedly large number of transcription factor binding site (TFBS) regions, with a minimal estimate of 12,000 for Sp1, 25,000 for cMyc, and 1600 for p53 when extrapolated to the full genome. Only 22% of these TFBS regions are located at the 5' termini of protein-coding genes while 36% lie within or immediately 3' to well-characterized genes and are significantly correlated with noncoding RNAs. A significant number of these noncoding RNAs are regulated in response to retinoic acid, and overlapping pairs of protein-coding and noncoding RNAs are often coregulated. Thus, the human genome contains roughly comparable numbers of protein-coding and noncoding genes that are bound by common transcription factors and regulated by common environmental signals.