A Transcriptional Activator Is Part of an SCF Ubiquitin Ligase to Control Degradation of Its Cofactors

Abstract

Summary Multi-subunit protein complexes pose a challenge to the coordinated regulation of individual components. We show how the yeast transactivating factor Met4 functions as a component of the SCFMet30 ubiquitin ligase to synchronize its own activity with co-factor assembly. Cells maintain Met4 in a dormant state by a regulatory ubiquitin chain assembled by SCFMet30. Nutritional and heavy metal stress block Met4 ubiquitylation resulting in Met4 activation, which induces a stress response program including cell cycle arrest. Met4 relies on assembly with various co-factors for promoter binding. We report here that the stability of these DNA-binding co-factors is regulated by SCFMet30. Remarkably, the transcriptional activator Met4 functions as a substrate specificity factor in the context of SCFMet30/Met4 to coordinate co-factor degradation with its own activity status. Our results establish an additional layer for substrate recruitment by SCF ubiquitin ligases, and provide conceptual insight into coordinated regulation of protein complexes.

Transcription Factor Condensates Mediate Clustering of MET Regulon and Enhancement in Gene Expression

“…To test these predictions, we first performed chromatin immunoprecipitation with sequencing (ChIP-seq) of related TFs (Met4, Met32, Met28, Cbf1), as well as RNA-seq, to identify genes directly targeted by Met4. As expected, the bindings of Met4, Met32, and Met28 are strongly induced by met depletion, while Cbf1, an abundant pioneer factor (Kuras et al 1996; Blaiseau and Thomas 1998; Ouni et al 2010; Carrillo et al 2012; Yan et al 2018; Donovan et al 2019), shows constitutive binding ( Fig. 3B & C, S3A & B ).…”

Transcription Factor Condensates Mediate Clustering of MET Regulon and Enhancement in Gene Expression

“…To test these predictions, we first performed chromatin immunoprecipitation with sequencing (ChIP-seq) of related TFs (Met4, Met32, Met28, Cbf1), as well as RNA-seq, to identify genes directly targeted by Met4. As expected, the bindings of Met4, Met32, and Met28 are strongly induced by met depletion, while Cbf1, an abundant pioneer factor (Kuras et al 1996; Blaiseau and Thomas 1998; Ouni et al 2010; Carrillo et al 2012; Yan et al 2018; Donovan et al 2019), shows constitutive binding ( Fig. 3B & C, S3A & B ).…”

Transcription factor condensates, 3D clustering, and gene expression enhancement of the MET regulon

“…To test these predictions, we first performed chromatin immunoprecipitation with sequencing (ChIP-seq) of related TFs (Met4, Met32, Met28, Cbf1), as well as RNA-seq, to identify genes directly targeted by Met4. As expected, the bindings of Met4, Met32, and Met28 are strongly induced by met depletion, while Cbf1, an abundant pioneer factor ( Kuras et al, 1996 ; Blaiseau and Thomas, 1998 ; Ouni et al, 2010 ; Carrillo et al, 2012 ; Yan et al, 2018 ; Donovan et al, 2019 ), shows constitutive binding ( Figure 3B, C , Figure 3—figure supplement 1A, B ). Under the induced condition, we identified 34 regions co-bound by all four TFs ( Figure 3C , Supplementary file 3 ), and these binding sites potentially regulate 46 genes (some sites are located in divergent promoters).…”

Transcription Activation Domains of the Yeast Factors Met4 and Ino2: Tandem Activation Domains with Properties Similar to the Yeast Gcn4 Activator

“…The deletion analysis also found that Met4 residues 161 to 168 repress AD function 40% to 50%. Part of this region is conserved and contains the ubiquitinated K163 residue (46,47). Western analysis results are consistent with modification at this residue, as this fusion protein migrated in a series of lower-mobility species in SDS-PAGE, although protein levels appeared unchanged from those seen with Met4 1-160 -Gcn4 ( Fig.…”

Transcription Factor Condensates Mediate Clustering of MET Regulon and Enhancement in Gene Expression

“…To test these predictions, we first performed chromatin immunoprecipitation with sequencing (ChIP-seq) of related TFs (Met4, Met32, Met28, Cbf1), as well as RNA-seq, to identify genes directly targeted by Met4. As expected, the bindings of Met4, Met32, and Met28 are strongly induced by met depletion, while Cbf1, an abundant pioneer factor (Kuras et al 1996; Blaiseau and Thomas 1998; Ouni et al 2010; Carrillo et al 2012; Yan et al 2018; Donovan et al 2019), shows constitutive binding ( Fig. 3B & C, S3A & B ).…”

Transcription factor condensates, 3D clustering, and gene expression enhancement of the MET regulon

“…To test these predictions, we first performed chromatin immunoprecipitation with sequencing (ChIP-seq) of related TFs (Met4, Met32, Met28, Cbf1), as well as RNA-seq, to identify genes directly targeted by Met4. As expected, the bindings of Met4, Met32, and Met28 are strongly induced by met depletion, while Cbf1, an abundant pioneer factor ( Kuras et al, 1996 ; Blaiseau and Thomas, 1998 ; Ouni et al, 2010 ; Carrillo et al, 2012 ; Yan et al, 2018 ; Donovan et al, 2019 ), shows constitutive binding ( Figure 3B, C , Figure 3—figure supplement 1A, B ). Under the induced condition, we identified 34 regions co-bound by all four TFs ( Figure 3C , Supplementary file 3 ), and these binding sites potentially regulate 46 genes (some sites are located in divergent promoters).…”

Transcription Activation Domains of the Yeast Factors Met4 and Ino2: Tandem Activation Domains with Properties Similar to the Yeast Gcn4 Activator

“…The deletion analysis also found that Met4 residues 161 to 168 repress AD function 40% to 50%. Part of this region is conserved and contains the ubiquitinated K163 residue (46,47). Western analysis results are consistent with modification at this residue, as this fusion protein migrated in a series of lower-mobility species in SDS-PAGE, although protein levels appeared unchanged from those seen with Met4 1-160 -Gcn4 ( Fig.…”

Transcription Factor Condensates Mediate Clustering of MET Regulon and Enhancement in Gene Expression

“…To test these predictions, we first performed chromatin immunoprecipitation with sequencing (ChIP-seq) of related TFs (Met4, Met32, Met28, Cbf1), as well as RNA-seq, to identify genes directly targeted by Met4. As expected, the bindings of Met4, Met32, and Met28 are strongly induced by met depletion, while Cbf1, an abundant pioneer factor (Kuras et al 1996; Blaiseau and Thomas 1998; Ouni et al 2010; Carrillo et al 2012; Yan et al 2018; Donovan et al 2019), shows constitutive binding ( Fig. 3B & C, S3A & B ).…”

Transcription factor condensates, 3D clustering, and gene expression enhancement of the MET regulon

“…To test these predictions, we first performed chromatin immunoprecipitation with sequencing (ChIP-seq) of related TFs (Met4, Met32, Met28, Cbf1), as well as RNA-seq, to identify genes directly targeted by Met4. As expected, the bindings of Met4, Met32, and Met28 are strongly induced by met depletion, while Cbf1, an abundant pioneer factor ( Kuras et al, 1996 ; Blaiseau and Thomas, 1998 ; Ouni et al, 2010 ; Carrillo et al, 2012 ; Yan et al, 2018 ; Donovan et al, 2019 ), shows constitutive binding ( Figure 3B, C , Figure 3—figure supplement 1A, B ). Under the induced condition, we identified 34 regions co-bound by all four TFs ( Figure 3C , Supplementary file 3 ), and these binding sites potentially regulate 46 genes (some sites are located in divergent promoters).…”

Transcription Activation Domains of the Yeast Factors Met4 and Ino2: Tandem Activation Domains with Properties Similar to the Yeast Gcn4 Activator

“…The deletion analysis also found that Met4 residues 161 to 168 repress AD function 40% to 50%. Part of this region is conserved and contains the ubiquitinated K163 residue (46,47). Western analysis results are consistent with modification at this residue, as this fusion protein migrated in a series of lower-mobility species in SDS-PAGE, although protein levels appeared unchanged from those seen with Met4 1-160 -Gcn4 ( Fig.…”