RESUMO
BACKGROUND: Van der Woude syndrome (VWS) is the most common form of syndromic orofacial cleft caused predominantly by mutations in Interferon Regulatory Factor 6 (IRF6). We previously reported that individuals with VWS have increased risk of wound healing complications following cleft repair compared with individuals with nonsyndromic orofacial clefts (nonsyndromic cleft lip and palate-NSCLP). In vitro, absence of IRF6 leads to impaired keratinocyte migration and embryonic wound healing. However, there is currently no data on tissue repair in adult animals and cells with reduced levels of IRF6 like in VWS. RESULTS: Excisional wounds of Irf6+/- and wild-type animals were analyzed 4 and 7 days post-wounding. Although all wounds were reepithelialized after 7 days, the epidermal and wound volume of repaired wounds was larger in Irf6+/- . These data were supported by increased keratinocyte proliferation in the neoformed epidermis and a less mature granulation tissue with increased cytokine levels. This effect was not cell autonomous, as Irf6+/- neonatal keratinocytes in vitro did not exhibit defects in scratch wound closure or proliferation. Keratinocytes from individuals with VWS also migrated similarly to keratinocytes from NSCLP individuals. CONCLUSIONS: These data support a role for IRF6 in wound healing by regulating keratinocyte proliferation, granulation tissue maturation, and cytokine levels.
Assuntos
Fatores Reguladores de Interferon/metabolismo , Cicatrização/fisiologia , Animais , Western Blotting , Ciclo Celular/genética , Ciclo Celular/fisiologia , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Células Cultivadas , Citocinas/metabolismo , Humanos , Fatores Reguladores de Interferon/genética , Queratinócitos/metabolismo , Camundongos Endogâmicos C57BL , Mutação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Cicatrização/genéticaRESUMO
Transcription by RNA polymerase II (Pol II) in eukaryotes requires the Mediator complex, and often involves chromatin remodeling and histone eviction at active promoters. Here we address the role of Mediator in recruitment of the Swi/Snf chromatin remodeling complex and its role, along with components of the preinitiation complex (PIC), in histone eviction at inducible and constitutively active promoters in the budding yeast Saccharomyces cerevisiae. We show that recruitment of the Swi/Snf chromatin remodeling complex to the induced CHA1 promoter, as well as its association with several constitutively active promoters, depends on the Mediator complex but is independent of Mediator at the induced MET2 and MET6 genes. Although transcriptional activation and histone eviction at CHA1 depends on Swi/Snf, Swi/Snf recruitment is not sufficient for histone eviction at the induced CHA1 promoter. Loss of Swi/Snf activity does not affect histone occupancy of several constitutively active promoters; in contrast, higher histone occupancy is seen at these promoters in Mediator and PIC component mutants. We propose that an initial activator-dependent, nucleosome remodeling step allows PIC components to outcompete histones for occupancy of promoter sequences. We also observe reduced promoter association of Mediator and TATA-binding protein in a Pol II (rpb1-1) mutant, indicating mutually cooperative binding of these components of the transcription machinery and indicating that it is the PIC as a whole whose binding results in stable histone eviction.
Assuntos
Histonas/metabolismo , Complexo Mediador/metabolismo , Regiões Promotoras Genéticas/genética , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína de Ligação a TATA-Box/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Northern Blotting , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Imunoprecipitação da Cromatina , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Complexo Mediador/genética , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Ligação Proteica , RNA Polimerase II/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteína de Ligação a TATA-Box/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Iniciação da Transcrição Genética , Ativação TranscricionalRESUMO
BACKGROUND: RhoA GTPase plays critical roles in actin cytoskeletal remodeling required for controlling a diverse range of cellular functions including cell proliferation, cell adhesions, migration and changes in cell shape. RhoA cycles between an active GTP-bound and an inactive GDP-bound form, a process that is regulated by guanine nucleotide exchange factors (GEFs), and GTPase-activating proteins (GAPs). ARHGAP29 is a GAP expressed in keratinocytes of the skin and is decreased in the absence of Interferon Regulator Factor 6, a critical regulator of cell proliferation and migration. However, the role for ARHGAP29 in keratinocyte biology is unknown. RESULTS: Novel ARHGAP29 knockdown keratinocyte cell lines were generated using both CRISPR/Cas9 and shRNA technologies. Knockdown cells exhibited significant reduction of ARHGAP29 protein (50-80%) and displayed increased filamentous actin (stress fibers), phospho-myosin light chain (contractility), cell area and population doubling time. Furthermore, we found that ARHGAP29 knockdown keratinocytes displayed significant delays in scratch wound closure in both single cell and collective cell migration conditions. Particularly, our results show a reduction in path lengths, speed, directionality and persistence in keratinocytes with reduced ARHGAP29. The delay in scratch closure was rescued by both adding back ARHGAP29 or adding a ROCK inhibitor to ARHGAP29 knockdown cells. CONCLUSIONS: These data demonstrate that ARHGAP29 is required for keratinocyte morphology, proliferation and migration mediated through the RhoA pathway.