SETDB1 deletion causes DNA demethylation and upregulation of multiple zinc-finger genes.

BACKGROUND: SETDB1 (SET domain bifurcated-1) is a histone H3-lysine 9 (H3K9)-specific methyltransferase that mediates heterochromatin formation and repression of target genes. Despite the assumed functional link between DNA methylation and SETDB1-mediated H3K9 trimethylations, several studies have shown that SETDB1 operates autonomously of DNA methylation in a region- and cell-specific manner. This study analyzes SETDB1-null HAP1 cells through a linked methylome and transcriptome analysis, intending to explore genes controlled by SETDB1-involved DNA methylation. METHODS AND RESULTS: We investigated SETDB1-mediated regulation of DNA methylation and gene transcription in human HAP1 cells using reduced-representation bisulfite sequencing (RRBS) and RNA sequencing. While two-thirds of differentially methylated CpGs (DMCs) in genic regions were hypomethylated in SETDB1-null cells, we detected a plethora of C2H2-type zinc-finger protein genes (C2H2-ZFP, 223 of 749) among the DMC-associated genes. Most C2H2-ZFPs with DMCs in their promoters were found hypomethylated in SETDB1-KO cells, while other non-ZFP genes with promoter DMCs were not. These C2H2-ZFPs with DMCs in their promoters were significantly upregulated in SETDB1-KO cells. Similarly, C2H2-ZFP genes were upregulated in SETDB1-null 293T cells, suggesting that SETDB1's function in ZFP gene repression is widespread. There are several C2H2-ZFP gene clusters on chromosome 19, which were selectively hypomethylated in SETDB1-KO cells. CONCLUSIONS: SETDB1 collectively and specifically represses a substantial fraction of the C2H2-ZFP gene family. Through the en-bloc silencing of a set of ZFP genes, SETDB1 may help establish a panel of ZFP proteins that are expressed cell-type specifically and thereby can serve as signature proteins for cellular identity.

Coordinated regulation of microRNA genes in C19MC by SETDB1.

The microRNA (miRNA) gene cluster on chromosome 19, C19MC, is the largest primate-specific miRNA gene cluster. The 46 homologous miRNA genes in C19MC are highly expressed in the placenta, but repressed in other tissues by DNA methylation. Here, we found that the SET domain bifurcated 1(SETDB1), a histone H3-lysine 9 (H3K9)-specific methyltransferase 1, transcriptionally controls C19MC miRNA genes in a coordinated manner in human HAP1 cells. SETDB1 knockout (KO) resulted in the overexpression of C19MC miRNA genes, which was accompanied by a reduction of H3K9 trimethylation (H3K9me3) in the cluster. We found that SETDB1 specifically binds to and modifies the upstream promoter locus of C19MC with H3K9me3, suggesting its role as a C19MC repressor. Overexpression of C19MC miRNA genes was not related to DNA methylation because cytosine methylation levels were not altered in the C19MC of SETDB1 KO cells, indicating that SETDB1 KO does not cause DNA demethylation in the C19MC promoter and body regions. In conclusion, our results suggest that SETDB1 binding and H3K9 methylation at the C19MC promoter and body regions are responsible for the coordinated regulation of miRNA genes in the cluster.

Different phases of aging in mouse old skeletal muscle.

With a graying population and increasing longevity, it is essential to identify life transition in later years and discern heterogeneity among older people. Subclassifying the elderly population to inspect the subdivisions for pathophysiological differences is particularly important for the investigation of age-related illnesses. For this purpose, using 24- and 28-month-old mice to represent the "young-old" and "old-old", respectively, we compared their skeletal muscle transcriptomes and found each in a distinct stage: early/gradual (E-aging) and late/accelerated aging phase (L-aging). Principal component analysis showed that the old-old transcriptomes were largely disengaged from the forward transcriptomic trajectory generated in the younger-aged group, indicating a substantial change in gene expression profiles during L-aging. By calculating the transcriptomic distance, it was found that the 28-month group was closer to the two-month group than to the 24-month group. The divergence rate per month for the transcriptomes was the highest in L-aging, twice as fast as the rate in E-aging. Indeed, many of the L-aging genes were significantly altered in transcription, although the changes did not seem random but rather coordinated in a variety of functional gene sets. Of 2,707 genes transcriptionally altered during E-aging, two-thirds were also significantly changed during L-aging, to either downturning or upturning way. The downturn genes were related to mitochondrial function and translational gene sets, while the upturn genes were linked to inflammation-associated gene sets. Our results provide a transcriptomic muscle signature that distinguishes old-old mice from young-old mice. This can help to methodically examine muscle disorders in the elderly.

Functional dissection of N-terminal nuclear trafficking signals of SETDB1.

SETDB1 is a histone H3-lysine 9-specific methyltransferase that fulfills epigenetic functions inside the nucleus; however, when overexpressed, SETDB1 majorily localizes in the cytoplasm. SETDB1 has a single nuclear-localization-signal (NLS) motif and two successive nuclear-export-signal (NES1 and NES2) motifs in the N-terminus, suggesting that SETDB1 localization is the consequence of a balance between the two antithetic motifs. Here, we performed a series of motif deletions to characterize their effects on the cellular movement of SETDB1. Given the cytoplasmic localization of GFP-SETDB1 in the whole form, without the NES motifs, GFP-SETDB1 was not nuclear, and 3xNLS addition plus NES removal held the majority of GFP-SETDB1 within the nucleus. The results indicated that the cytoplasmic localization of GFP-SETDB1 is the combined result of weak NLS and robust NESs. In ATF7IP-overexpressing cells, GFP-SETDB1 entered the nucleus only in the presence of the NES1 motif; neither the NES2 nor NLS motif was necessary. Since subcellular fractionation results showed that ATF7IP was nuclear-only, an intermediary protein may interact specifically with the NES1 motif after stimulation by ATF7IP. When GFP-SETDB1 had either NES1 or NES2, it was precipitated (in immunoprecipitation) and colocalized (in immunofluorescence) with ATF7IP, indicating that GFP-SETDB1 interacts with ATF7IP through the NES motifs in the nucleus. The regulated nuclear entry of SETDB1 is assumed to set a tight restriction on its abundance within the nucleus, thereby ensuring balanced nuclear SETDB1 levels.

Customization Methodology for Conformable Grasping Posture of Soft Grippers by Stiffness Patterning.

Soft grippers with soft and flexible materials have been widely researched to improve the functionality of grasping. Although grippers that can grasp various objects with different shapes are important, a large number of industrial applications require a gripper that is targeted for a specified object. In this paper, we propose a design methodology for soft grippers that are customized to grasp single dedicated objects. A customized soft gripper can safely and efficiently grasp a dedicated target object with lowered surface contact forces while maintaining a higher lifting force, compared to its non-customized counterpart. A simplified analytical model and a fabrication method that can rapidly customize and fabricate soft grippers are proposed. Stiffness patterns were implemented onto the constraint layers of pneumatic bending actuators to establish actuated postures with irregular bending curvatures in the longitudinal direction. Soft grippers with customized stiffness patterns yielded higher shape conformability to target objects than non-patterned regular soft grippers. The simplified analytical model represents the pneumatically actuated soft finger as a summation of interactions between its air chambers. Geometric approximations and pseudo-rigid-body modeling theory were employed to build the analytical model. The customized soft grippers were compared with non-patterned soft grippers by measuring their lifting forces and contact forces while they grasped objects. Under the identical actuating pressure, the conformable grasping postures enabled customized soft grippers to have almost three times the lifting force than that of non-patterned soft grippers, while the maximum contact force was reduced to two thirds.