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1.
Nat Commun ; 15(1): 3110, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38600112

RESUMO

Homeodomains (HDs) are the second largest class of DNA binding domains (DBDs) among eukaryotic sequence-specific transcription factors (TFs) and are the TF structural class with the largest number of disease-associated mutations in the Human Gene Mutation Database (HGMD). Despite numerous structural studies and large-scale analyses of HD DNA binding specificity, HD-DNA recognition is still not fully understood. Here, we analyze 92 human HD mutants, including disease-associated variants and variants of uncertain significance (VUS), for their effects on DNA binding activity. Many of the variants alter DNA binding affinity and/or specificity. Detailed biochemical analysis and structural modeling identifies 14 previously unknown specificity-determining positions, 5 of which do not contact DNA. The same missense substitution at analogous positions within different HDs often exhibits different effects on DNA binding activity. Variant effect prediction tools perform moderately well in distinguishing variants with altered DNA binding affinity, but poorly in identifying those with altered binding specificity. Our results highlight the need for biochemical assays of TF coding variants and prioritize dozens of variants for further investigations into their pathogenicity and the development of clinical diagnostics and precision therapies.


Assuntos
Proteínas de Homeodomínio , Fatores de Transcrição , Humanos , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , DNA/metabolismo , Mutação , Modelos Moleculares
2.
Stem Cell Reports ; 18(6): 1325-1339, 2023 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-37315524

RESUMO

Skeletal muscle function and regenerative capacity decline during aging, yet factors driving these changes are incompletely understood. Muscle regeneration requires temporally coordinated transcriptional programs to drive myogenic stem cells to activate, proliferate, fuse to form myofibers, and to mature as myonuclei, restoring muscle function after injury. We assessed global changes in myogenic transcription programs distinguishing muscle regeneration in aged mice from young mice by comparing pseudotime trajectories from single-nucleus RNA sequencing of myogenic nuclei. Aging-specific differences in coordinating myogenic transcription programs necessary for restoring muscle function occur following muscle injury, likely contributing to compromised regeneration in aged mice. Differences in pseudotime alignment of myogenic nuclei when comparing aged with young mice via dynamic time warping revealed pseudotemporal differences becoming progressively more severe as regeneration proceeds. Disruptions in timing of myogenic gene expression programs may contribute to incomplete skeletal muscle regeneration and declines in muscle function as organisms age.


Assuntos
Núcleo Celular , Células-Tronco , Animais , Camundongos , Envelhecimento/genética , Músculo Esquelético , Expressão Gênica
3.
Cell Rep ; 34(1): 108574, 2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33406418

RESUMO

The zinc finger transcription factor SALL4 is highly expressed in embryonic stem cells, downregulated in most adult tissues, but reactivated in many aggressive cancers. This unique expression pattern makes SALL4 an attractive therapeutic target. However, whether SALL4 binds DNA directly to regulate gene expression is unclear, and many of its targets in cancer cells remain elusive. Here, through an unbiased screen of protein binding microarray (PBM) and cleavage under targets and release using nuclease (CUT&RUN) experiments, we identify and validate the DNA binding domain of SALL4 and its consensus binding sequence. Combined with RNA sequencing (RNA-seq) analyses after SALL4 knockdown, we discover hundreds of new SALL4 target genes that it directly regulates in aggressive liver cancer cells, including genes encoding a family of histone 3 lysine 9-specific demethylases (KDMs). Taken together, these results elucidate the mechanism of SALL4 DNA binding and reveal pathways and molecules to target in SALL4-dependent tumors.


Assuntos
Carcinoma Hepatocelular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Regulação Neoplásica da Expressão Gênica , Histona Desmetilases/metabolismo , Fatores de Transcrição/metabolismo , Dedos de Zinco , Motivos de Aminoácidos , Sequência de Aminoácidos , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Histona Desmetilases/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Análise Serial de Proteínas , Ligação Proteica , Análise de Sequência de RNA , Fatores de Transcrição/genética
4.
Mol Cell ; 77(2): 324-337.e8, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31704182

RESUMO

A major challenge in biology is to understand how complex gene expression patterns are encoded in the genome. While transcriptional enhancers have been studied extensively, few transcriptional silencers have been identified, and they remain poorly understood. Here, we used a novel strategy to screen hundreds of sequences for tissue-specific silencer activity in whole Drosophila embryos. Almost all of the transcriptional silencers that we identified were also active enhancers in other cellular contexts. These elements are bound by more transcription factors than non-silencers. A subset of these silencers forms long-range contacts with promoters. Deletion of a silencer caused derepression of its target gene. Our results challenge the common practice of treating enhancers and silencers as separate classes of regulatory elements and suggest the possibility that thousands or more bifunctional CRMs remain to be discovered in Drosophila and 104-105 in humans.


Assuntos
Drosophila/genética , Elementos Facilitadores Genéticos/genética , Elementos Silenciadores Transcricionais/genética , Transcrição Gênica/genética , Animais , Animais Geneticamente Modificados/genética , Masculino
5.
Cell ; 173(2): 430-442.e17, 2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29606353

RESUMO

Fetal hemoglobin (HbF, α2γ2) level is genetically controlled and modifies severity of adult hemoglobin (HbA, α2ß2) disorders, sickle cell disease, and ß-thalassemia. Common genetic variation affects expression of BCL11A, a regulator of HbF silencing. To uncover how BCL11A supports the developmental switch from γ- to ß- globin, we use a functional assay and protein binding microarray to establish a requirement for a zinc-finger cluster in BCL11A in repression and identify a preferred DNA recognition sequence. This motif appears in embryonic and fetal-expressed globin promoters and is duplicated in γ-globin promoters. The more distal of the duplicated motifs is mutated in individuals with hereditary persistence of HbF. Using the CUT&RUN approach to map protein binding sites in erythroid cells, we demonstrate BCL11A occupancy preferentially at the distal motif, which can be disrupted by editing the promoter. Our findings reveal that direct γ-globin gene promoter repression by BCL11A underlies hemoglobin switching.


Assuntos
Proteínas de Transporte/metabolismo , Hemoglobina Fetal/genética , Proteínas Nucleares/metabolismo , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte/genética , Linhagem Celular , Cromatina/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Células Eritroides/citologia , Células Eritroides/metabolismo , Edição de Genes , Humanos , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Repressoras , Dedos de Zinco/genética , Globinas beta/genética , Talassemia beta/genética , Talassemia beta/patologia , gama-Globinas/genética
6.
Cell Rep ; 22(12): 3227-3239, 2018 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-29562179

RESUMO

Little is known about how variation in sequence composition alters transcription factor occupancy to precisely recruit large transcription complexes. A key model for understanding how transcription complexes are targeted is the Drosophila dosage compensation system in which the male-specific lethal (MSL) transcription complex specifically identifies and regulates the male X chromosome. The chromatin-linked adaptor for MSL proteins (CLAMP) zinc-finger protein targets MSL to the X chromosome but also binds to GA-rich sequence elements throughout the genome. Furthermore, the GAGA-associated factor (GAF) transcription factor also recognizes GA-rich sequences but does not associate with the MSL complex. Here, we demonstrate that MSL complex recruitment sites are optimal CLAMP targets. Specificity for CLAMP binding versus GAF binding is driven by variability in sequence composition within similar GA-rich motifs. Therefore, variation within seemingly similar cis elements drives the context-specific targeting of a large transcription complex.


Assuntos
Drosophila/genética , Drosophila/metabolismo , Animais , Feminino , Fator de Transcrição de Proteínas de Ligação GA/metabolismo , Masculino , Cromossomos Sexuais , Cromossomo X
7.
Science ; 351(6280): 1450-1454, 2016 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-27013732

RESUMO

Sequencing of exomes and genomes has revealed abundant genetic variation affecting the coding sequences of human transcription factors (TFs), but the consequences of such variation remain largely unexplored. We developed a computational, structure-based approach to evaluate TF variants for their impact on DNA binding activity and used universal protein-binding microarrays to assay sequence-specific DNA binding activity across 41 reference and 117 variant alleles found in individuals of diverse ancestries and families with Mendelian diseases. We found 77 variants in 28 genes that affect DNA binding affinity or specificity and identified thousands of rare alleles likely to alter the DNA binding activity of human sequence-specific TFs. Our results suggest that most individuals have unique repertoires of TF DNA binding activities, which may contribute to phenotypic variation.


Assuntos
Proteínas de Ligação a DNA/genética , DNA/metabolismo , Regulação da Expressão Gênica , Doenças Genéticas Inatas/genética , Fatores de Transcrição/genética , Sequência de Bases , Sítios de Ligação , Simulação por Computador , Proteínas de Ligação a DNA/metabolismo , Exoma/genética , Variação Genética , Genoma Humano , Humanos , Mutação , Polimorfismo de Nucleotídeo Único , Análise Serial de Proteínas , Ligação Proteica , Análise de Sequência de DNA , Fatores de Transcrição/metabolismo
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