Biomedical subjects

Juhong Liu

Publications and source records attributed to Juhong Liu.

4 recordsLinked to original sources

PubMed2006

The FUSE/FBP/FIR/TFIIH system is a molecular machine programming a pulse of c-myc expression.

FarUpStream Element (FUSE) Binding Protein (FBP) binds the human c-myc FUSE in vitro only in single-stranded or supercoiled DNA. Because transcriptionally generated torsion melts FUSE in vitro even in linear DNA, and FBP/FBP Interacting Repressor (FIR) regulates transcription through TFIIH, these components have been speculated to be the mechanosensor (FUSE) and effectors (FBP/FIR) of a real-time mechanism controlling c-myc transcription. To ascertain whether the FUSE/FBP/FIR system operates according to this hypothesis in vivo, the flux of activators, repressors and chromatin remodeling complexes on the c-myc promoter was monitored throughout the serum-induced pulse of transcription. After transcription was switched on by conventional factors and chromatin regulators, FBP and FIR were recruited and established a dynamically remodeled loop with TFIIH at the P2 promoter. In XPB cells carrying mutant TFIIH, loop formation failed and the serum response was abnormal; RNAi depletion of FIR similarly disabled c-myc regulation. Engineering FUSE into episomal vectors predictably re-programmed metallothionein-promoter-driven reporter expression. The in vitro recruitment of FBP and FIR to dynamically stressed c-myc DNA paralleled the in vivo process.

Cell Line↗

PubMed2006

FBPs are calibrated molecular tools to adjust gene expression.

The three far-upstream element (FUSE) binding protein (FBP) family members have been ascribed different functions in gene regulation. They were therefore examined with various biochemical, molecular biological, and cell biological tests to evaluate whether their sequence differences reflect functional customization or neutral changes at unselected residues. Each FBP displayed a characteristic profile of intrinsic transcription activation and repression, binding with protein partners, and subcellular trafficking. Although some differences, such as weakened FBP3 nuclear localization, were predictable from primary sequence differences, the unexpected failure of FBP3 to bind the FBP-interacting repressor (FIR) was traced to seemingly conservative substitutions within a small patch of an N-terminal alpha-helix. The transactivation strength and the FIR-binding strength of the FBPs were in the opposite order. Despite their distinguishing features and differential activities, the FBPs traffic to shared subnuclear sites and regulate many common target genes, including c-myc. Though a variety of functions have been attributed to the FBPs, based upon their panel of shared and unique features, we propose that they constitute a molecular regulatory kit that tunes the expression of shared targets through a common mechanism.

Amino Acid Sequence↗

PubMed2005

TFIIH operates through an expanded proximal promoter to fine-tune c-myc expression.

A continuous stream of activating and repressing signals is processed by the transcription complex paused at the promoter of the c-myc proto-oncogene. The general transcription factor IIH (TFIIH) is held at promoters prior to promoter escape and so is well situated to channel the input of activators and repressors to modulate c-myc expression. We have compared cells expressing only a mutated p89 (xeroderma pigmentosum complementation group B [XPB]), the largest TFIIH subunit, with the same cells functionally complemented with the wild-type protein (XPB/wt-p89). Here, we show structural, compositional, and functional differences in transcription complexes between XPB and XPB/wt-89 cells at the native c-myc promoter. Remarkably, although the mean levels of c-Myc are only modestly elevated in XPB compared to those in XPB/wt-p89 cells, the range of expression and the cell-to-cell variation of c-Myc are markedly increased. Our modeling indicates that the data can be explained if TFIIH integrates inputs from multiple signals, regulating transcription at multiple kinetically equivalent steps between initiation and promoter escape. This helps to suppress the intrinsic noise of transcription and to ensure the steady transcriptional output of c-myc necessary for cellular homeostasis.

Blotting, Western↗

PubMed2004

The dynamic response of upstream DNA to transcription-generated torsional stress.

The torsional stress caused by counter-rotation of the transcription machinery and template generates supercoils in a closed topological domain, but has been presumed to be too short-lived to be significant in an open domain. This report shows that transcribing RNA polymerases dynamically sustain sufficient torsion to perturb DNA structure even on linear templates. Assays to capture and measure transcriptionally generated torque and to trap short-lived perturbations in DNA structure and conformation showed that the transient forces upstream of active promoters are large enough to drive the supercoil-sensitive far upstream element (FUSE) of the human c-myc into single-stranded DNA. An alternative non-B conformation of FUSE found in stably supercoiled DNA is not accessible dynamically. These results demonstrate that dynamic disturbance of DNA structure provides a real-time measure of ongoing genetic activity.

Base Sequence↗