Person:

Liu, Jessica

Seamless and minimally-invasive integration of three-dimensional (3D) electronic circuitry within host materials could enable the development of materials systems that are self- monitoring and allow for communication with external environments. Here, we report a general strategy for preparing ordered 3D interconnected and addressable macroporous nanoelectronic networks from ordered two-dimensional (2D) nanowire nanoelectronic “precursors”, which are fabricated by conventional lithography. The 3D networks have porosities larger than 99%, contain ca. 100’s of addressable nanowire devices, and have feature sizes from the 10 micron scale (for electrical and structural interconnections) to the 10 nanometer scale (for device elements). The macroporous nanoelectronic networks were merged with organic gels and polymers to form hybrid materials in which the basic physical and chemical properties of the host were not substantially altered, and electrical measurements further show a > 90% yield of active devices in the hybrid materials. The positions of the nanowire devices were located within 3D hybrid materials with ca. 14 nm resolution through simultaneous nanowire device photocurrent/confocal microscopy imaging measurements. In addition, we explored functional properties of these hybrid materials, including (i) mapping time-dependent pH changes throughout a nanowire network/agarose gel sample during external solution pH changes, and (ii) characterizing the strain field in a hybrid nanoelectronic elastomer structures subject to uniaxial and bending forces. The seamless incorporation of active nanoelectronic networks within 3D materials opens up a powerful approach to smart materials in which the capabilities of multi- functional nanoelectronics allow for active monitoring and control of host systems.

The sites where the basic unit of chromatin, the nucleosome, is assembled greatly affects the dynamic compaction/decompaction of eukaryotic genetic material and how the DNA is accessed, read, and interpreted. The nucleosome, which consists of ~147 base pairs of DNA wrapped in a left-handed superhelix around an octameric core made up of histone proteins, is the targeted substrate for ATP-dependent protein machineries called chromatin remodelers. Remodelers are essential regulators of DNA accessibility and are often grouped into four families: SWI/SNF, INO80/SWR1, ISWI, and CHD. Though remodelers can act as large multi-subunit complexes, all have a unique core SNF2-like ATPase that utilizes the energy from ATP hydrolysis to translocate along DNA. This DNA translocase activity of the catalytic ATPase domain acts in coordination with auxiliary domains or accessory subunits to disrupt histone-DNA contacts, resulting in distinct remodeling outcomes. Furthermore, the assembly of DNA into nucleosomal arrays is a specialized activity catalyzed by a subset of remodelers. Identifying remodeler proteins responsible for nucleosome assembly and delineating the mechanisms through which remodelers assemble and remodel nucleosomes are key goals in the field of chromatin biology.
CHD proteins have important roles in regulating gene expression through their remodeling activities. While yeast cells only have one CHD protein (CHD1), mammalians possess nine proteins (CHD1-9) that are further categorized into subfamilies on the basis of additional sequences flanking the central ATPase domain. CHD2 is in the same subfamily as CHD1 and has been linked to developmental regulation but the enzymatic activity of CHD2 has not been well characterized. Given the homology between human CHD2 and CHD1, which is an important assembly protein in other species (S. cerevisiae and D. melanogaster), we set out to delineate the biochemical properties of human CHD2 and the CHD1 human counterpart. In this dissertation work, we examined the biochemical activities of recombinant human CHD1 and CHD2. We used in vitro chromatin assembly and remodeling assays and showed CHD2 assembles nucleosomal arrays and remodels nucleosomes while CHD1 exhibits less robust activity by comparison. We used radiometric ATPase and electrophoretic mobility gel shift assays to measure the ATPase and DNA-binding activities of human CHD1 and CHD2 and assessed the contribution from conserved accessory domains using systematic protein truncations. We found the N-terminal chromodomains are inhibitory for the ATPase and DNA-binding activities of both CHD1 and CHD2 while providing substrate specificity for the latter. Moreover, we showed the DNA-binding domain of CHD2 enhances its ATPase and remodeling activities. The distinct in vitro activities exhibited by human CHD1 and CHD2 suggest they have non-redundant roles in vivo with important mechanistic implications for remodeling by CHD proteins. In a broader sense, our findings have added to the number of known assembly motor proteins and aids in our understanding of how remodelers have evolved auxiliary domains to carry out specific functions such as chromatin assembly.

Abstract CHD1 is a conserved chromatin remodeling enzyme required for development and linked to prostate cancer in adults, yet its role in human cells is poorly understood. Here, we show that targeted disruption of the CHD1 gene in human cells leads to a defect in early double-strand break (DSB) repair via homologous recombination (HR), resulting in hypersensitivity to ionizing radiation as well as PARP and PTEN inhibition. CHD1 knockout cells show reduced H2AX phosphorylation (γH2AX) and foci formation as well as impairments in CtIP recruitment to the damaged sites. Chromatin immunoprecipitation following a single DSB shows that the reduced levels of γH2AX accumulation at DSBs in CHD1-KO cells are due to both a global reduction in H2AX incorporation and poor retention of H2AX at the DSBs. We also identified a unique N-terminal region of CHD1 that inhibits the DNA binding, ATPase, and chromatin assembly and remodeling activities of CHD1. CHD1 lacking the N terminus was more active in rescuing the defects in γH2AX formation and CtIP recruitment in CHD1-KO cells than full-length CHD1, suggesting the N terminus is a negative regulator in cells. Our data point to a role for CHD1 in the DSB repair process and identify a novel regulatory region of the protein.