Myc and SAGA rewire an alternative splicing network during early somatic cell reprogramming.

Embryonic stem cells are maintained in a self-renewing and pluripotent state by multiple regulatory pathways. Pluripotent-specific transcriptional networks are sequentially reactivated as somatic cells reprogram to achieve pluripotency. How epigenetic regulators modulate this process and contribute to somatic cell reprogramming is not clear. Here we performed a functional RNAi screen to identify the earliest epigenetic regulators required for reprogramming. We identified components of the SAGA histone acetyltransferase complex, in particular Gcn5, as critical regulators of reprogramming initiation. Furthermore, we showed in mouse pluripotent stem cells that Gcn5 strongly associates with Myc and that, upon initiation of somatic reprogramming, Gcn5 and Myc form a positive feed-forward loop that activates a distinct alternative splicing network and the early acquisition of pluripotency-associated splicing events. These studies expose a Myc-SAGA pathway that drives expression of an essential alternative splicing regulatory network during somatic cell reprogramming.

MBNL proteins repress ES-cell-specific alternative splicing and reprogramming.

Previous investigations of the core gene regulatory circuitry that controls the pluripotency of embryonic stem (ES) cells have largely focused on the roles of transcription, chromatin and non-coding RNA regulators. Alternative splicing represents a widely acting mode of gene regulation, yet its role in regulating ES-cell pluripotency and differentiation is poorly understood. Here we identify the muscleblind-like RNA binding proteins, MBNL1 and MBNL2, as conserved and direct negative regulators of a large program of cassette exon alternative splicing events that are differentially regulated between ES cells and other cell types. Knockdown of MBNL proteins in differentiated cells causes switching to an ES-cell-like alternative splicing pattern for approximately half of these events, whereas overexpression of MBNL proteins in ES cells promotes differentiated-cell-like alternative splicing patterns. Among the MBNL-regulated events is an ES-cell-specific alternative splicing switch in the forkhead family transcription factor FOXP1 that controls pluripotency. Consistent with a central and negative regulatory role for MBNL proteins in pluripotency, their knockdown significantly enhances the expression of key pluripotency genes and the formation of induced pluripotent stem cells during somatic cell reprogramming.

Absolute scaling of single-cell transcriptomes identifies pervasive hypertranscription in adult stem and progenitor cells.

Hypertranscription supports biosynthetically demanding cellular states through global transcriptome upregulation. Despite its potential widespread relevance, documented examples of hypertranscription remain few and limited to early development. Here, we demonstrate that absolute scaling of single-cell RNA-sequencing data enables the estimation of total transcript abundances per cell. We validate absolute scaling in known cases of developmental hypertranscription and apply it to adult cell types, revealing a remarkable dynamic range in transcriptional output. In adult organs, hypertranscription marks activated stem/progenitor cells with multilineage potential and is redeployed in conditions of tissue injury, where it precedes bursts of proliferation during regeneration. Our analyses identify a common set of molecular pathways associated with both adult and embryonic hypertranscription, including chromatin remodeling, DNA repair, ribosome biogenesis, and translation. These shared features across diverse cell contexts support hypertranscription as a general and dynamic cellular program that is pervasively employed during development, organ maintenance, and regeneration.

A late transition in somatic cell reprogramming requires regulators distinct from the pluripotency network.

Reprogramming of somatic cells to a pluripotent state via expression of Oct4, Klf4, Myc, and Sox2 is a multistep process involving phased changes in gene expression. Here, we focus on the later stages of reprogramming, termed maturation and stabilization. We show that the stabilization phase and the acquisition of pluripotency are dependent on the removal of transgene expression late in the maturation phase. Clonal analysis of cells undergoing reprogramming revealed subsets of stabilization-competent (SC) and stabilization-incompetent (SI) cells. SC clones acquire a competency gene-expression signature late in the maturation phase. Functional analysis of SC signature genes identified enhancers of the transition to the stabilization phase and a distinct subset of genes required for the maintenance of pluripotency. Thus, the acquisition and maintenance of pluripotency are regulated by distinct molecular networks, and a specific regulatory program not previously implicated in reprogramming is required for the transition to transgene independence.

Collagen stimulates discoidin domain receptor 1-mediated migration of smooth muscle cells through Src.

BACKGROUND: Discoidin domain receptor 1 (DDR1) is a collagen-binding receptor tyrosine kinase which mediates the migration and proliferation of several cell types. DDR1 is expressed in vascular smooth muscle cells (SMCs) during atherosclerosis and following vascular injury, mediating cell migration and contributing to disease pathogenesis. However, very little is known about the signaling pathways activated by the DDR1 in SMCs. Therefore we have studied the involvement of Src and mitogen-activated protein kinase (MAPK) signaling pathways downstream of DDR1 in vascular SMCs. METHODS: Cells harvested from DDR1(-/-), DDR1(+/+) mice, and DDR1(+/+) cells overexpressing human DDR1b (O/hDDR1b) were used for these studies. RESULTS: Stimulation of O/hDDR1b cells with type I collagen resulted in increased tyrosine phosphorylation of DDR1. The non-receptor kinase Src co-immunoprecipitated with DDR1, and the Src inhibitor PP2 inhibited type I collagen-induced tyrosine phosphorylation of DDR1. Stimulation of DDR1-expressing cells with collagen resulted in the activation of extracellular signal-regulated kinase 1/2 (ERK1/2); however, ERK1/2 was not activated in DDR1-deficient cells. By contrast, p38 MAPK (p38) was activated by collagen stimulation in both DDR1-expressing and DDR1-deficient cells. Treatment with PP2 attenuated DDR1-dependent ERK1/2 activation, but not p38 activation. Finally, treatment of SMCs with PP2, or the MEK inhibitor PD98059, inhibited migration toward type I collagen in a chemotaxis chamber. However, PP2 but not PD98059 had a greater effect in reducing the migration of DDR1(+/+) cells compared to DDR1(-/-) cells, suggesting that Src but not ERK1/2 was important in regulating DDR1-dependent SMC migration. CONCLUSIONS: Type I collagen induces SMC migration through DDR1 and this is mediated via Src signaling.