AP-1 is a temporally regulated dual gatekeeper of reprogramming to pluripotency.

Somatic cell transcription factors are critical to maintaining cellular identity and constitute a barrier to human somatic cell reprogramming; yet a comprehensive understanding of the mechanism of action is lacking. To gain insight, we examined epigenome remodeling at the onset of human nuclear reprogramming by profiling human fibroblasts after fusion with murine embryonic stem cells (ESCs). By assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and chromatin immunoprecipitation sequencing we identified enrichment for the activator protein 1 (AP-1) transcription factor c-Jun at regions of early transient accessibility at fibroblast-specific enhancers. Expression of a dominant negative AP-1 mutant (dnAP-1) reduced accessibility and expression of fibroblast genes, overcoming the barrier to reprogramming. Remarkably, efficient reprogramming of human fibroblasts to induced pluripotent stem cells was achieved by transduction with vectors expressing SOX2, KLF4, and inducible dnAP-1, demonstrating that dnAP-1 can substitute for exogenous human OCT4. Mechanistically, we show that the AP-1 component c-Jun has two unexpected temporally distinct functions in human reprogramming: 1) to potentiate fibroblast enhancer accessibility and fibroblast-specific gene expression, and 2) to bind to and repress OCT4 as a complex with MBD3. Our findings highlight AP-1 as a previously unrecognized potent dual gatekeeper of the somatic cell state.

HoxC4 binds to the promoter of the cytidine deaminase AID gene to induce AID expression, class-switch DNA recombination and somatic hypermutation.

The cytidine deaminase AID (encoded by Aicda in mice and AICDA in humans) is critical for immunoglobulin class-switch recombination (CSR) and somatic hypermutation (SHM). Here we show that AID expression was induced by the HoxC4 homeodomain transcription factor, which bound to a highly conserved HoxC4-Oct site in the Aicda or AICDA promoter. This site functioned in synergy with a conserved binding site for the transcription factors Sp1, Sp3 and NF-kappaB. HoxC4 was 'preferentially' expressed in germinal center B cells and was upregulated by engagement of CD40 by CD154, as well as by lipopolysaccharide and interleukin 4. HoxC4 deficiency resulted in impaired CSR and SHM because of lower AID expression and not some other putative HoxC4-dependent activity. Enforced expression of AID in Hoxc4(-/-) B cells fully restored CSR. Thus, HoxC4 directly activates the Aicda promoter, thereby inducing AID expression, CSR and SHM.

Induction of activation-induced cytidine deaminase-targeting adaptor 14-3-3γ is mediated by NF-κB-dependent recruitment of CFP1 to the 5'-CpG-3'-rich 14-3-3γ promoter and is sustained by E2A.

Class switch DNA recombination (CSR) crucially diversifies Ab biologic effector functions. 14-3-3γ specifically binds to the 5'-AGCT-3' repeats in the IgH locus switch (S) regions. By interacting directly with the C-terminal region of activation-induced cytidine deaminase (AID), 14-3-3γ targets this enzyme to S regions to mediate CSR. In this study, we showed that 14-3-3γ was expressed in germinal center B cells in vivo and induced in B cells by T-dependent and T-independent primary CSR-inducing stimuli in vitro in humans and mice. Induction of 14-3-3γ was rapid, peaking within 3 h of stimulation by LPSs, and sustained over the course of AID and CSR induction. It was dependent on recruitment of NF-κB to the 14-3-3γ gene promoter. The NF-κB recruitment enhanced the occupancy of the CpG island within the 14-3-3γ promoter by CFP1, a component of the COMPASS histone methyltransferase complex, and promoter-specific enrichment of histone 3 lysine 4 trimethylation (H3K4me3), which is indicative of open chromatin state and marks transcription-competent promoters. NF-κB also potentiated the binding of B cell lineage-specific factor E2A to an E-box motif located immediately downstream of the two closely-spaced transcription start sites for sustained 14-3-3γ expression and CSR induction. Thus, 14-3-3γ induction in CSR is enabled by the CFP1-mediated H3K4me3 enrichment in the promoter, dependent on NF-κB and sustained by E2A.

Elevated CD47 is a hallmark of dysfunctional aged muscle stem cells that can be targeted to augment regeneration.

In aging, skeletal muscle strength and regenerative capacity decline, due in part to functional impairment of muscle stem cells (MuSCs), yet the underlying mechanisms remain elusive. Here, we capitalize on mass cytometry to identify high CD47 expression as a hallmark of dysfunctional MuSCs (CD47hi) with impaired regenerative capacity that predominate with aging. The prevalent CD47hi MuSC subset suppresses the residual functional CD47lo MuSC subset through a paracrine signaling loop, leading to impaired proliferation. We uncover that elevated CD47 levels on aged MuSCs result from increased U1 snRNA expression, which disrupts alternative polyadenylation. The deficit in aged MuSC function in regeneration can be overcome either by morpholino-mediated blockade of CD47 alternative polyadenylation or antibody blockade of thrombospondin-1/CD47 signaling, leading to improved regeneration in aged mice, with therapeutic implications. Our findings highlight a previously unrecognized age-dependent alteration in CD47 levels and function in MuSCs, which underlies reduced muscle repair in aging.

Estrogen receptors bind to and activate the HOXC4/HoxC4 promoter to potentiate HoxC4-mediated activation-induced cytosine deaminase induction, immunoglobulin class switch DNA recombination, and somatic hypermutation.

Estrogen enhances antibody and autoantibody responses through yet to be defined mechanisms. It has been suggested that estrogen up-regulates the expression of activation-induced cytosine deaminase (AID), which is critical for antibody class switch DNA recombination (CSR) and somatic hypermutation (SHM), through direct activation of this gene. AID, as we have shown, is induced by the HoxC4 homeodomain transcription factor, which binds to a conserved HoxC4/Oct site in the AICDA/Aicda promoter. Here we show that estrogen-estrogen receptor (ER) complexes do not directly activate the AID gene promoter in B cells undergoing CSR. Rather, they bind to three evolutionarily conserved and cooperative estrogen response elements (EREs) we identified in the HOXC4/HoxC4 promoter. By binding to these EREs, ERs synergized with CD154 or LPS and IL-4 signaling to up-regulate HoxC4 expression, thereby inducing AID and CSR without affecting B cell proliferation or plasmacytoid differentiation. Estrogen administration in vivo significantly potentiated CSR and SHM in the specific antibody response to the 4-hydroxy-3-nitrophenylacetyl hapten conjugated with chicken γ-globulin. Ablation of HoxC4 (HoxC4(-/-)) abrogated the estrogen-mediated enhancement of AID gene expression and decreased CSR and SHM. Thus, estrogen enhances AID expression by activating the HOXC4/HoxC4 promoter and inducing the critical AID gene activator, HoxC4.

Glucose Metabolism Drives Histone Acetylation Landscape Transitions that Dictate Muscle Stem Cell Function.

The impact of glucose metabolism on muscle regeneration remains unresolved. We identify glucose metabolism as a crucial driver of histone acetylation and myogenic cell fate. We use single-cell mass cytometry (CyTOF) and flow cytometry to characterize the histone acetylation and metabolic states of quiescent, activated, and differentiating muscle stem cells (MuSCs). We find glucose is dispensable for mitochondrial respiration in proliferating MuSCs, so that glucose becomes available for maintaining high histone acetylation via acetyl-CoA. Conversely, quiescent and differentiating MuSCs increase glucose utilization for respiration and have consequently reduced acetylation. Pyruvate dehydrogenase (PDH) activity serves as a rheostat for histone acetylation and must be controlled for muscle regeneration. Increased PDH activity in proliferation increases histone acetylation and chromatin accessibility at genes that must be silenced for differentiation to proceed, and thus promotes self-renewal. These results highlight metabolism as a determinant of MuSC histone acetylation, fate, and function during muscle regeneration.

NKX3-1 is required for induced pluripotent stem cell reprogramming and can replace OCT4 in mouse and human iPSC induction.

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) is now routinely accomplished by overexpression of the four Yamanaka factors (OCT4, SOX2, KLF4, MYC (or OSKM))1. These iPSCs can be derived from patients' somatic cells and differentiated toward diverse fates, serving as a resource for basic and translational research. However, mechanistic insights into regulators and pathways that initiate the pluripotency network remain to be resolved. In particular, naturally occurring molecules that activate endogenous OCT4 and replace exogenous OCT4 in human iPSC reprogramming have yet to be found. Using a heterokaryon reprogramming system we identified NKX3-1 as an early and transiently expressed homeobox transcription factor. Following knockdown of NKX3-1, iPSC reprogramming is abrogated. NKX3-1 functions downstream of the IL-6-STAT3 regulatory network to activate endogenous OCT4. Importantly, NKX3-1 substitutes for exogenous OCT4 to reprogram both mouse and human fibroblasts at comparable efficiencies and generate fully pluripotent stem cells. Our findings establish an essential role for NKX3-1, a prostate-specific tumour suppressor, in iPSC reprogramming.

Publisher Correction: High-resolution myogenic lineage mapping by single-cell mass cytometry.

In the version of this Article originally published, the name of author Andrew Tri Van Ho was coded wrongly, resulting in it being incorrect when exported to citation databases. This has been corrected, though no visible changes will be apparent.

High-resolution myogenic lineage mapping by single-cell mass cytometry.

Muscle regeneration is a dynamic process during which cell state and identity change over time. A major roadblock has been a lack of tools to resolve a myogenic progression in vivo. Here we capitalize on a transformative technology, single-cell mass cytometry (CyTOF), to identify in vivo skeletal muscle stem cell and previously unrecognized progenitor populations that precede differentiation. We discovered two cell surface markers, CD9 and CD104, whose combined expression enabled in vivo identification and prospective isolation of stem and progenitor cells. Data analysis using the X-shift algorithm paired with single-cell force-directed layout visualization defined a molecular signature of the activated stem cell state (CD44+/CD98+/MyoD+) and delineated a myogenic trajectory during recovery from acute muscle injury. Our studies uncover the dynamics of skeletal muscle regeneration in vivo and pave the way for the elucidation of the regulatory networks that underlie cell-state transitions in muscle diseases and ageing.

Immunoglobulin class-switch DNA recombination: induction, targeting and beyond.

Class-switch DNA recombination (CSR) of the immunoglobulin heavy chain (IGH) locus is central to the maturation of the antibody response and crucially requires the cytidine deaminase AID. CSR involves changes in the chromatin state and the transcriptional activation of the IGH locus at the upstream and downstream switch (S) regions that are to undergo S-S DNA recombination. In addition, CSR involves the induction of AID expression and the targeting of CSR factors to S regions by 14-3-3 adaptors, and it is facilitated by the transcription machinery and by histone modifications. In this Review, we focus on recent advances regarding the induction and targeting of CSR and outline an integrated model of the assembly of macromolecular complexes that transduce crucial epigenetic information to enzymatic effectors of the CSR machinery.

Rev1 recruits ung to switch regions and enhances du glycosylation for immunoglobulin class switch DNA recombination.

By diversifying the biological effector functions of antibodies, class switch DNA recombination (CSR) plays a critical role in the maturation of the immune response. It is initiated by activation-induced cytidine deaminase (AID)-mediated deoxycytosine deamination, yielding deoxyuridine (dU), and dU glycosylation by uracil DNA glycosylase (Ung) in antibody switch (S) region DNA. Here we showed that the translesion DNA synthesis polymerase Rev1 directly interacted with Ung and targeted in an AID-dependent and Ung-independent fashion the S regions undergoing CSR. Rev1(-/-)Ung(+/+) B cells reduced Ung recruitment to S regions, DNA-dU glycosylation, and CSR. Together with an S region spectrum of mutations similar to that of Rev1(+/+)Ung(-/-) B cells, this suggests that Rev1 operates in the same pathway as Ung, as emphasized by further decreased CSR in Rev1(-/-)Msh2(-/-) B cells. Rescue of CSR in Rev1(-/-) B cells by a catalytically inactive Rev1 mutant shows that the important role of Rev1 in CSR is mediated by Rev1's scaffolding function, not its enzymatic function.

BCR-signalling synergizes with TLR-signalling for induction of AID and immunoglobulin class-switching through the non-canonical NF-κB pathway.

By diversifying antibody biological effector functions, class switch DNA recombination has a central role in the maturation of the antibody response. Here we show that BCR-signalling synergizes with Toll-like receptor (TLR) signalling to induce class switch DNA recombination. BCR-signalling activates the non-canonical NF-κB pathway and enhances the TLR-dependent canonical NF-κB pathway, thereby inducing activation-induced cytidine deaminase (AID), which is critical for class switch DNA recombination. Escherichia coli lipopolysaccharide (LPS) triggers dual TLR4/BCR-signalling and induces hallmarks of BCR-signalling, including CD79a phosphorylation and Ca(2+) mobilization, and activates both the NF-κB pathways to induce AID and class switch DNA recombination in a PI(3)K p85α-dependent fashion. CD40-signalling activates the two NF-κB pathways to induce AID and class switch DNA recombination independent of BCR-signalling. Finally, dual BCR/TLR-engaging NP-lipopolysaccharide effectively elicits class-switched NP-specific IgG3 and IgG2b in mice. Thus, by integrating signals of the non-canonical and canonical NF-κB pathways, BCR and TLRs synergize to induce AID and T-cell-independent class switch DNA recombination.

Endonuclease G plays a role in immunoglobulin class switch DNA recombination by introducing double-strand breaks in switch regions.

Immunoglobulin (Ig) class switch DNA recombination (CSR) is the crucial mechanism diversifying the biological effector functions of antibodies. Generation of double-strand DNA breaks (DSBs), particularly staggered DSBs, in switch (S) regions of the upstream and downstream CH genes involved in the specific recombination process is an absolute requirement for CSR. Staggered DSBs would be generated through deamination of dCs on opposite DNA strands by activation-induced cytidine deaminase (AID), subsequent dU deglycosylation by uracil DNA glycosylase (Ung) and abasic site nicking by apurinic/apyrimidic endonuclease. However, consistent with the findings that significant amounts of DSBs can be detected in the IgH locus in the absence of AID or Ung, we have shown in human and mouse B cells that AID generates staggered DSBs not only by cleaving intact double-strand DNA, but also by processing blunt DSB ends generated in an AID-independent fashion. How these AID-independent DSBs are generated is still unclear. It is possible that S region DNA may undergo AID-independent cleavage by structure-specific nucleases, such as endonuclease G (EndoG). EndoG is an abundant nuclease in eukaryotic cells. It cleaves single and double-strand DNA, primarily at dG/dC residues, the preferential sites of DSBs in S region DNA. We show here that EndoG can localize to the nucleus of B cells undergoing CSR and binds to S region DNA, as shown by specific chromatin immunoprecipitation assays. Using knockout EndoG(-/-) mice and EndoG(-/-) B cells, we found that EndoG deficiency resulted in a two-fold reduction in CSR in vivo and in vitro, as demonstrated by reduced cell surface IgG1, IgG2a, IgG3 and IgA, reduced secreted IgG1, reduced circle Iγ1-Cµ, Iγ3-Cµ, Iɛ-Cµ, Iα-Cµ transcripts, post-recombination Iµ-Cγ1, Iµ-Cγ3, Iµ-Cɛ and Iµ-Cα transcripts. In addition to reduced CSR, EndoG(-/-) mice showed a significantly altered spectrum of mutations in IgH J(H)-iEµ DNA. Impaired CSR in EndoG(-/-) B cells did not stem from altered B cell proliferation or apoptosis. Rather, it was associated with significantly reduced frequency of DSBs. Thus, our findings determine a role for EndoG in the generation of S region DSBs and CSR.

AID dysregulation in lupus-prone MRL/Fas(lpr/lpr) mice increases class switch DNA recombination and promotes interchromosomal c-Myc/IgH loci translocations: modulation by HoxC4.

Immunoglobulin gene somatic hypermutation (SHM) and class switch DNA recombination (CSR) play important roles in the generation of autoantibodies in systemic lupus erythematosus. Systemic lupus is characterized by the production of an array of pathogenic high-affinity mutated and class-switched, mainly IgG, antibodies to a variety of self-antigens, including nuclear components, such as dsDNA, histones, and chromatin. We previously found that MRL/Fas(lpr/lpr) mice, which develop a systemic autoimmune syndrome sharing many features with human lupus, display greatly upregulated CSR, particularly to IgG2a, in B cells of the spleen, lymph nodes, and Peyer's patches. In MRL/Fas(lpr/lpr) mice, the significant upregulation of CSR is associated with increased expression of activation-induced cytidine deaminase (AID), which is critical for CSR and SHM. We also found that HoxC4 directly activates the promoter of the AID gene to induce AID expression, CSR and SHM. Here, we show that in both lupus patients and lupus-prone MRL/Fas(lpr/lpr) mice, the expression of HoxC4 and AID is significantly upregulated. To further analyze the role of HoxC4 in lupus, we generated HoxC4(-/-) MRL/Fas(lpr/lpr) mice. In these mice, HoxC4-deficiency resulted in reduced AID expression, impaired CSR, and decreased serum anti-dsDNA IgG, particularly IgG2a, autoantibodies, which were associated with a reduction in IgG deposition in kidney glomeruli. In addition, consistent with our previous findings in MRL/Fas(lpr/lpr) mice that upregulated AID expression is associated with extensive DNA lesions, comprising deletions and insertions in the IgH locus, we found that c-Myc to IgH (c-Myc/IgH) translocations occur frequently in B cells of MRL/Fas(lpr/lpr) mice. The frequency of such translocations was significantly reduced in HoxC4(-/-) MRL/Fas(lpr/lpr) mice. These findings suggest that in lupus B cells, upregulation of HoxC4 plays a major role in dysregulation of AID expression, thereby increasing CSR and autoantibody production and promoting c-Myc/IgH translocations.

14-3-3 adaptor proteins recruit AID to 5'-AGCT-3'-rich switch regions for class switch recombination.

Class switch DNA recombination (CSR) is the mechanism that diversifies the biological effector functions of antibodies. Activation-induced cytidine deaminase (AID), a key protein in CSR, targets immunoglobulin H (IgH) switch regions, which contain 5'-AGCT-3' repeats in their core. How AID is recruited to switch regions remains unclear. Here we show that 14-3-3 adaptor proteins have an important role in CSR. 14-3-3 proteins specifically bound 5'-AGCT-3' repeats, were upregulated in B cells undergoing CSR and were recruited with AID to the switch regions that are involved in CSR events (Smu-->Sgamma1, Smu-->Sgamma3 or Smu-->Salpha). Moreover, blocking 14-3-3 by difopein, 14-3-3gamma deficiency or expression of a dominant-negative 14-3-3sigma mutant impaired recruitment of AID to switch regions and decreased CSR. Finally, 14-3-3 proteins interacted directly with AID and enhanced AID-mediated in vitro DNA deamination, further emphasizing the important role of these adaptors in CSR.