Transcription is a process finely regulated by different transcription factors (TFs) which bind regulatory sequences present in gene promoters and allow the precise execution of gene expression programs. Misregulation of such process can lead to different pathologies, including development/differentiation defects, uncontrolled cell growth and cancer. For these reasons it is important to understand the molecular details of the interplay that occurs between different TFs to modulate gene expression. NF-Y, the heterotrimeric complex composed by NF-YA, NF-YB and NF-YC subunits, all required for DNA binding, recognizes the consensus sequence CCAAT, present in about 30% of eukaryotic promoters, at -60/-100 bp from the Transcription Start Site (TSS). One of the most important roles of NF-Y in transcription is to interact synergistically with other TFs to activate, or to repress, gene expression. In this study we focused on the relationship occurring between NF-Y and the TFs MAX, Myc and USF1, which recognize the E-BOX sequence CACGTG. We made this choice relying on genomic information and ChIP-seq data derived from the ENCODE project. These studies revealed that in CCAAT promoters, several Transcription Factor Binding Sites (TFBS) are consistently present. Among these, the E-BOX consensus is one of the most frequently found, generally at a distance from CCAAT of 10/12 bp, in a precise orientation; moreover CCAAT and E-BOXes, at these distances, are bound in vivo by NF-Y an E-BOX TFs, respectively. In addition to genomic information, the knowledge of NF-Y and E-BOX TFs domains structures in complex with DNA allows us to speculate on the possible interactions that might occur when DNA, with this configuration, is bound, and bent, by the two proteins. Therefore, we analyzed and characterized the relationships among NF-Y and MAX, Myc or USF1 using Electrophoretic Mobility Shift Assay (EMSA) and ChIP analyses: in vitro EMSAs were performed using E-BOX TFs (MAX or USF1 as homodimers, and Myc/MAX as heterodimers) and NF-Y proteins purified from E. coli; as probes, DNA sequences derived from genomic loci that reflect the observed distances and orientation of CCAAT and E-BOX. In particular, we chose DNA sequences derived from the HOXB4 and HOXB7 gene promoters, and the ERV9 Long Terminal Repeat (LTR) region. With these experiments, we found that NF-Y can form stable ternary complexes with all recombinant E-BOX TFs and DNA configurations analyzed, with no negative interactions. We observed that USF1 and NF-Y display a facilitated binding on HOXB4, that becomes even more stable on HOXB7, and cooperative on the LTR sequence. Moreover, we observed that the minimal domains of NF-Y and USF1 maintain these DNA binding features. ChIP experiments in HeLa cells, previously silenced for NF-Y, revealed that the interplay between NF-Y and E-BOX TFs exists also in vivo. In fact, after NF-Y silencing, Myc, MAX and USF1 binding is affected, directly or indirectly, further supporting the notion that NF-Y and E-BOX TFs cooperate in the regulation of target genes that contain CCAAT and E-BOXes in their promoters.

THE INTERPLAY AMONG NF-Y AND E-BOX TRANSCRIPTION FACTORS: MAX, MYC AND USF1 / M. Lorenzo ; scientific tutor: R. Mantovani, N. Gnesutta. - : . DIPARTIMENTO DI BIOSCIENZE, 2015 May 22. ((27. ciclo, Anno Accademico 2014. [10.13130/lorenzo-mariangela_phd2015-05-22].

THE INTERPLAY AMONG NF-Y AND E-BOX TRANSCRIPTION FACTORS: MAX, MYC AND USF1

M. Lorenzo
2015-05-22

Abstract

Transcription is a process finely regulated by different transcription factors (TFs) which bind regulatory sequences present in gene promoters and allow the precise execution of gene expression programs. Misregulation of such process can lead to different pathologies, including development/differentiation defects, uncontrolled cell growth and cancer. For these reasons it is important to understand the molecular details of the interplay that occurs between different TFs to modulate gene expression. NF-Y, the heterotrimeric complex composed by NF-YA, NF-YB and NF-YC subunits, all required for DNA binding, recognizes the consensus sequence CCAAT, present in about 30% of eukaryotic promoters, at -60/-100 bp from the Transcription Start Site (TSS). One of the most important roles of NF-Y in transcription is to interact synergistically with other TFs to activate, or to repress, gene expression. In this study we focused on the relationship occurring between NF-Y and the TFs MAX, Myc and USF1, which recognize the E-BOX sequence CACGTG. We made this choice relying on genomic information and ChIP-seq data derived from the ENCODE project. These studies revealed that in CCAAT promoters, several Transcription Factor Binding Sites (TFBS) are consistently present. Among these, the E-BOX consensus is one of the most frequently found, generally at a distance from CCAAT of 10/12 bp, in a precise orientation; moreover CCAAT and E-BOXes, at these distances, are bound in vivo by NF-Y an E-BOX TFs, respectively. In addition to genomic information, the knowledge of NF-Y and E-BOX TFs domains structures in complex with DNA allows us to speculate on the possible interactions that might occur when DNA, with this configuration, is bound, and bent, by the two proteins. Therefore, we analyzed and characterized the relationships among NF-Y and MAX, Myc or USF1 using Electrophoretic Mobility Shift Assay (EMSA) and ChIP analyses: in vitro EMSAs were performed using E-BOX TFs (MAX or USF1 as homodimers, and Myc/MAX as heterodimers) and NF-Y proteins purified from E. coli; as probes, DNA sequences derived from genomic loci that reflect the observed distances and orientation of CCAAT and E-BOX. In particular, we chose DNA sequences derived from the HOXB4 and HOXB7 gene promoters, and the ERV9 Long Terminal Repeat (LTR) region. With these experiments, we found that NF-Y can form stable ternary complexes with all recombinant E-BOX TFs and DNA configurations analyzed, with no negative interactions. We observed that USF1 and NF-Y display a facilitated binding on HOXB4, that becomes even more stable on HOXB7, and cooperative on the LTR sequence. Moreover, we observed that the minimal domains of NF-Y and USF1 maintain these DNA binding features. ChIP experiments in HeLa cells, previously silenced for NF-Y, revealed that the interplay between NF-Y and E-BOX TFs exists also in vivo. In fact, after NF-Y silencing, Myc, MAX and USF1 binding is affected, directly or indirectly, further supporting the notion that NF-Y and E-BOX TFs cooperate in the regulation of target genes that contain CCAAT and E-BOXes in their promoters.
MANTOVANI, ROBERTO
Transcription Factors; NF-Y; E-BOX; interplay
Settore BIO/10 - Biochimica
Settore BIO/11 - Biologia Molecolare
THE INTERPLAY AMONG NF-Y AND E-BOX TRANSCRIPTION FACTORS: MAX, MYC AND USF1 / M. Lorenzo ; scientific tutor: R. Mantovani, N. Gnesutta. - : . DIPARTIMENTO DI BIOSCIENZE, 2015 May 22. ((27. ciclo, Anno Accademico 2014. [10.13130/lorenzo-mariangela_phd2015-05-22].
Doctoral Thesis
File in questo prodotto:
File Dimensione Formato  
phd_unimi_R09767.pdf

embargo fino al 30/10/2015

Tipologia: Tesi di dottorato completa
Dimensione 4.77 MB
Formato Adobe PDF
4.77 MB Adobe PDF Visualizza/Apri
Pubblicazioni consigliate

Caricamento pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/274117
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact