Biomedical subjects

Danwei Huangfu

Publications and source records attributed to Danwei Huangfu.

7 recordsLinked to original sources

PubMed2025

A chemical epigenetic tool to probe site-specific DNA-binding protein complexes.

Site-specific DNA binding by proteins is critical for regulating transcriptional activity and cell fate decision. However, identifying proteins bound to specific genomic regions (e.g., promoter or enhancer regions) remains challenging. To address this, we developed a chemical epigenetic tool, named Site-specific noncanonical amino acid-mediated capture of protein (SCOPE), incorporating a photo-crosslinking amino acid into a nuclease-deficient dCas9 mutant. Human pluripotent stem cells (hPSCs) carrying SCOPE enable the capture of proteins bound to, in theory, any genomic location, facilitating the study of the cell context-dependent DNA-protein interactions. Using SCOPE, we identified the OCT4/SOX2/CARHSP1 complex binding to the NANOG promoter to maintain pluripotency in hPSCs. During ectoderm differentiation, ZIC2 acts as a competitive inhibitor, binding the same promoter region to downregulate NANOG expression and promote differentiation. Additionally, SCOPE identified that ZNF8 binds to the distal regulatory region of OCT4 to maintain naïve pluripotency. In summary, SCOPE provides a robust system for uncovering cell context-dependent, site-specific genome regulators, offering valuable insights into gene regulation networks driving cell fate transitions.

Humans↗

PubMed2025

Decoding heterogeneous single-cell perturbation responses.

Understanding how cells respond differently to perturbation is crucial in cell biology, but existing methods often fail to accurately quantify and interpret heterogeneous single-cell responses. Here we introduce the perturbation-response score (PS), a method to quantify diverse perturbation responses at a single-cell level. Applied to single-cell perturbation datasets such as Perturb-seq, PS outperforms existing methods in quantifying partial gene perturbations. PS further enables single-cell dosage analysis without needing to titrate perturbations, and identifies 'buffered' and 'sensitive' response patterns of essential genes, depending on whether their moderate perturbations lead to strong downstream effects. PS reveals differential cellular responses on perturbing key genes in contexts such as T cell stimulation, latent HIV-1 expression and pancreatic differentiation. Notably, we identified a previously unknown role for the coiled-coil domain containing 6 (CCDC6) in regulating liver and pancreatic cell fate decisions. PS provides a powerful method for dose-to-function analysis, offering deeper insights from single-cell perturbation data.

Single-Cell Analysis↗

PubMed2006

Signaling from Smo to Ci/Gli: conservation and divergence of Hedgehog pathways from Drosophila to vertebrates.

Although the framework of the Hedgehog (Hh) signaling pathway is evolutionarily conserved, recent studies indicate that fundamental differences exist between Drosophila and vertebrates in the way signals are transduced from the membrane protein Smoothened (Smo) to the Ci/Gli transcription factors. For example, Smo structure and the roles of fused and Suppressor of fused have diverged. Recently, many vertebrate-specific components have been identified that act between Smo and Gli. These include intra-flagellar transport proteins, which link vertebrate Hh signaling to cilia. Because abnormal Hh signaling can cause birth defects and cancer, these vertebrate-specific components may have roles in human health.

Animals↗

PubMed2005

Cilia and Hedgehog responsiveness in the mouse.

The intraflagellar transport (IFT) proteins Ift172/Wimple and Polaris/Ift88 and the anterograde IFT motor kinesin-II are required for the production and maintenance of cilia. These proteins are also required for the activation of targets of the mouse Hedgehog (Hh) pathway by Gli transcription factors. The phenotypes of the IFT mutants, however, are not identical to mutants that lack Smoothened (Smo), an essential activator of the Hh pathway. We show here that mouse embryos that lack both Ift172 and Smo are identical to Ift172 single mutants, which indicates that Ift172 acts downstream of Smo. Ift172 mutants have a weaker neural patterning phenotype than Smo mutants, because Ift172, but not Smo, is required for proteolytic processing of Gli3 to its repressor form. Dnchc2 and Kif3a, essential subunits of the retrograde and anterograde IFT motors, are also required for both formation of Gli activator and proteolytic processing of Gli3. As a result, IFT mutants display a loss of Hh signaling phenotype in the neural tube, where Gli activators play the major role in pattern formation, and a gain of Hh signaling phenotype in the limb, where Gli3 repressor plays the major role. Because both anterograde and retrograde IFT are essential for positive and negative responses to Hh, and because cilia are present on Hh responsive cells, it is likely that cilia act as organelles that are required for all activity of the mouse Hh pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Analysis of mouse embryonic patterning and morphogenesis by forward genetics.

Many aspects of the genetic control of mammalian embryogenesis cannot be extrapolated from other animals. Taking a forward genetic approach, we have induced recessive mutations by treatment of mice with ethylnitrosourea and have identified 43 mutations that affect early morphogenesis and patterning, including 38 genes that have not been studied previously. The molecular lesions responsible for 14 mutations were identified, including mutations in nine genes that had not been characterized previously. Some mutations affect vertebrate-specific components of conserved signaling pathways; for example, at least five mutations affect previously uncharacterized regulators of the Sonic hedgehog (Shh) pathway. Approximately half of all of the mutations affect the initial establishment of the body plan, and several of these produce phenotypes that have not been described previously. A large fraction of the genes identified affect cell migration, cellular organization, and cell structure. The findings indicate that phenotype-based genetic screens provide a direct and unbiased method to identify essential regulators of mammalian development.

Animals↗

PubMed2003

Hedgehog signalling in the mouse requires intraflagellar transport proteins.

Intraflagellar transport (IFT) proteins were first identified as essential factors for the growth and maintenance of flagella in the single-celled alga Chlamydomonas reinhardtii. In a screen for embryonic patterning mutations induced by ethylnitrosourea, here we identify two mouse mutants, wimple (wim) and flexo (fxo), that lack ventral neural cell types and show other phenotypes characteristic of defects in Sonic hedgehog signalling. Both mutations disrupt IFT proteins: the wim mutation is an allele of the previously uncharacterized mouse homologue of IFT172; and fxo is a new hypomorphic allele of polaris, the mouse homologue of IFT88. Genetic analysis shows that Wim, Polaris and the IFT motor protein Kif3a are required for Hedgehog signalling at a step downstream of Patched1 (the Hedgehog receptor) and upstream of direct targets of Hedgehog signalling. Our data show that IFT machinery has an essential and vertebrate-specific role in Hedgehog signal transduction.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Mouse Dispatched homolog1 is required for long-range, but not juxtacrine, Hh signaling.

Precise patterning of cell types along the dorsal-ventral axis of the spinal cord is essential to establish functional neural circuits. In order to prove the feasibility of studying a single biological process through random mutagenesis in the mouse, we have identified recessive ENU-induced mutations in six genes that prevent normal specification of ventral cell types in the spinal cord. We positionally cloned the genes responsible for two of the mutant phenotypes, smoothened and dispatched, which are homologs of Drosophila Hh pathway components. The Dispatched homolog1 (Disp1) mutation causes lethality at midgestation and prevents specification of ventral cell types in the neural tube, a phenotype identical to the Smoothened (Smo) null phenotype. As in Drosophila, mouse Disp1 is required to move Shh away from the site of synthesis. Despite the existence of a second mouse disp homolog, Disp1 is essential for long-range signaling by both Shh and Ihh ligands. Our data indicate that Shh signaling is required within the notochord to maintain Shh expression and to prevent notochord degeneration. Disp1, unlike Smo, is not required for this juxtacrine signaling by Shh.

Amino Acid Sequence↗