From the Cover: PNAS Plus: The CentO satellite confers translational and rotational phasing on cenH3 nucleosomes in rice centromeres

Plant and animal centromeres comprise megabases of highly repeated satellite sequences, yet centromere function can be specified epigenetically on single-copy DNA by the presence of nucleosomes containing a centromere-specific variant of histone H3 (cenH3). We determined the positions of cenH3 nucleosomes in rice (Oryza sativa), which has centromeres composed of both the 155-bp CentO satellite repeat and single-copy non-CentO sequences. We find that cenH3 nucleosomes protect 90–100 bp of DNA from micrococcal nuclease digestion, sufficient for only a single wrap of DNA around the cenH3 nucleosome core. cenH3 nucleosomes are translationally phased with 155-bp periodicity on CentO repeats, but not on non-CentO sequences. CentO repeats have an ∼10-bp periodicity in WW dinucleotides and in micrococcal nuclease cleavage, providing evidence for rotational phasing of cenH3 nucleosomes on CentO and suggesting that satellites evolve for translational and rotational stabilization of centromeric nucleosomes.Centromeres, the chromosomal domains that attach to spindle microtubules to segregate eukaryotic chromosomes in mitosis and meiosis, are DNA elements bound by special nucleosomes that contain a centromere-specific variant of histone H3 (cenH3). In most plants and animals, cenH3 nucleosomes are found on centromeric DNA that comprises megabases of tandemly repeated “satellite” sequences. Despite this apparent preference for repetitive DNA, a fully functional centromere, called a neocentromere, can occasionally form by assembling cenH3 nucleosomes on a single-copy DNA sequence that was not previously part of a centromere, indicating that centromere specification is epigenetic in plants and animals (for reviews, see refs. 1–4).The tandem arrays of highly repeated satellite sequences that compose most plant and animal centromeres can differ dramatically between closely related species (5), and even between different chromosomes (6–8), suggesting that satellite arrays undergo rapid evolution through expansions, contractions, gene conversions, and transpositions. Monomers of satellite repeats range in length from 5 bp in Drosophila to 1,419 bp in cattle although more than half of described monomers in 282 species have lengths between 100 and 200 bp, often regarded as approximately the length of nucleosomal DNA (6, 9). The cenH3 nucleosomes typically occupy only a portion of the satellite repeats, often in discontinuous blocks (7, 10–12), and the same or similar repeats often underlie flanking pericentromeric heterochromatin composed of conventional nucleosomes. Some of these repeats, for example African green monkey α-satellite DNA, have long been known to position conventional nucleosomes, resulting in arrays of regularly spaced nucleosomes, said to be translationally phased (13–15). Nucleosomes can occupy multiple alternative translational phases on the same satellite (16, 17). Translationally phased nucleosomal arrays have also been observed on satellites in cucumber and in several cereal species, where phasing varies among repeats and chromosomal regions (18, 19).Recently deep-sequencing technology has been applied to centromeres treated with micrococcal nuclease (MNase), which preferentially digests linker DNA between nucleosomes, to determine the positioning of cenH3 nucleosomes on satellite repeats. In human cultured cells, substantial translational phasing of CENP-A, the human cenH3, was reported on α-satellite (20). In maize, a similar approach mapped CENH3 (the name used for plant cenH3s) on the 156-bp maize centromeric satellite CentC and on two retrotransposon-derived centromeric sequences, CRM1 and CRM2 (21). Evidence for translational phasing of CENH3 on CentC and CRM1 was lacking, but 190-bp phasing was observed on CRM2. CentC was shown to have a strong periodicity of AA or TT dinucleotides about every 10 bp, which corresponds to one turn of the DNA double helix. This periodicity is thought to favor a particular orientation of the DNA toward the nucleosome core particle, based on DNA bendability, and is known as rotational phasing of nucleosomes (22–24).Rice has centromeres characterized by the 155-bp satellite sequence CentO, which is related to maize CentC (25, 26). Although some rice centromeres have megabases of CentO satellites, other evolutionarily new centromeres have little CentO, so CENH3 nucleosomes are found on both CentO and non-CentO sequences (12). For example, Cen8 is comprised of mostly non-CentO sequences and has a CentO array (CentO_8) that is spanned by a sequenced BAC (27). Centromeres like Cen8 are thought to represent an intermediate stage in centromere evolution between rare neocentromeres that form on unique sequences and mature centromeres populated by megabase-sized arrays of satellites (7, 12). Cen8 therefore presents an opportunity to compare the organization of CENH3 nucleosomes on CentO and non-CentO sequences. To that end, we used an antibody to rice CENH3 (27) to perform chromatin immunoprecipitation (ChIP) of CENH3 nucleosomes digested with MNase and sequenced the bound DNA (ChIP-Seq) to determine the positions of CENH3 nucleosomes on rice centromeres. We analyzed the sizes and positions of CENH3 nucleosomal DNA fragments on both CentO and non-CentO sequences to address the role of satellites in organizing centromeric chromatin and analyzed the sequence features of these fragments to look for evidence of nucleosome positioning signals.     

Structural insights into the histone H1-nucleosome complex

Linker H1 histones facilitate formation of higher-order chromatin structures and play important roles in various cell functions. Despite several decades of effort, the structural basis of how H1 interacts with the nucleosome remains elusive. Here, we investigated Drosophila H1 in complex with the nucleosome, using solution nuclear magnetic resonance spectroscopy and other biophysical methods. We found that the globular domain of H1 bridges the nucleosome core and one 10-base pair linker DNA asymmetrically, with its α3 helix facing the nucleosomal DNA near the dyad axis. Two short regions in the C-terminal tail of H1 and the C-terminal tail of one of the two H2A histones are also involved in the formation of the H1–nucleosome complex. Our results lead to a residue-specific structural model for the globular domain of the Drosophila H1 in complex with the nucleosome, which is different from all previous experiment-based models and has implications for chromatin dynamics in vivo.Eukaryotic genomic DNA is packaged into chromatin through association with positively charged histones to form the nucleosome, the structural unit of chromatin (1–3). The nucleosome core consists of an octamer of histones with two copies of H2A, H2B, H3, and H4, around which ∼146 bp of DNA winds in ∼1.65 left-handed superhelical turns (4). At this level of the DNA packaging, chromatin resembles a beads-on-a-string structure, with the nucleosome core as the beads and the linker DNA between them as the strings (5). At the next level of DNA packaging, H1 histones bind to the linker DNA and the nucleosome to further condense the chromatin structure (6, 7). H1-mediated chromatin condensation plays important roles in cellular functions such as mitotic chromosome architecture and segregation (8), muscle differentiation (9), and regulation of gene expression (10, 11).Linker H1 histones typically are ∼200 amino acid residues in length, with a short N-terminal region, followed by a ∼70–80-amino acid structured globular domain (gH1) and a ∼100-amino acid unstructured C-terminal domain that is highly enriched in Lys residues. H1 stabilizes the nucleosome and facilitates folding of nucleosome arrays into higher-order structures (12–15). gH1 alone confers the same protection from micrococcal nuclease digestion to the nucleosome as the full-length H1 does (16). The N-terminal region of H1 is not important for nucleosome binding (16, 17), whereas the C terminus is required for H1 binding to chromatin in vivo (18, 19) and for the formation of a stem structure of linker DNA in vitro (17, 20, 21).The globular domain structures of avian H5 (22) and budding yeast Hho1 (23), which are both H1 homologs, have been determined at atomic resolution and show similar structures. In addition, numerous studies have indicated that gH1/gH5 binds around the dyad region of the nucleosome (14, 24), leading to many conflicting structural models for how the globular domain of H1/H5 binds to the nucleosome (SI Appendix, Fig. S1) (24–26). These models are divided into two major classes, symmetric and asymmetric, on the basis of the location of gH1/gH5 in the nucleosome. In the symmetric class, gH1/gH5 binds to the nucleosomal DNA at the dyad and interacts with both linker DNAs (16, 17, 27, 28). In the asymmetric class, gH1/gH5 binds to the nucleosomal DNA in the vicinity of the dyad axis and to 10 bp (27, 29–32) or 20 bp (19, 29, 33, 34) of one linker DNA, or is located inside the DNA gyres, where it interacts with histone H2A (35). In addition, Zhou and colleagues also characterized the orientation of gH5 in the gH5-nucleosome complex (29). The use of nonuniquely positioned nucleosomes and indirect methods may have contributed to the differences in these models (SI Appendix, Fig. S1).Multidimensional nuclear magnetic resonance (NMR), and in particular methyl-based NMR, provides a direct approach to the structural characterization of macromolecular complexes (36, 37). We have previously assigned chemical shifts of the methyl groups of the side chains of residues Ile, Leu, and Val in the core histones (38) and the backbone amides in the disordered histone tails (39), which provide the fingerprints for investigating the interactions between H1 and the nucleosome. Here, we used NMR, along with several other methods, to determine the location and orientation of the globular domain of a stable mutant of Drosophila H1 on a well-positioned nucleosome.     

Histone chaperone Anp32e removes H2A.Z from DNA double-strand breaks and promotes nucleosome reorganization and DNA repair

The repair of DNA double-strand breaks (DSBs) requires open, flexible chromatin domains. The NuA4–Tip60 complex creates these flexible chromatin structures by exchanging histone H2A.Z onto nucleosomes and promoting acetylation of histone H4. Here, we demonstrate that the accumulation of H2A.Z on nucleosomes at DSBs is transient, and that rapid eviction of H2A.Z is required for DSB repair. Anp32e, an H2A.Z chaperone that interacts with the C-terminal docking domain of H2A.Z, is rapidly recruited to DSBs. Anp32e functions to remove H2A.Z from nucleosomes, so that H2A.Z levels return to basal within 10 min of DNA damage. Further, H2A.Z removal by Anp32e disrupts inhibitory interactions between the histone H4 tail and the nucleosome surface, facilitating increased acetylation of histone H4 following DNA damage. When H2A.Z removal by Anp32e is blocked, nucleosomes at DSBs retain elevated levels of H2A.Z, and assume a more stable, hypoacetylated conformation. Further, loss of Anp32e leads to increased CtIP-dependent end resection, accumulation of single-stranded DNA, and an increase in repair by the alternative nonhomologous end joining pathway. Exchange of H2A.Z onto the chromatin and subsequent rapid removal by Anp32e are therefore critical for creating open, acetylated nucleosome structures and for controlling end resection by CtIP. Dynamic modulation of H2A.Z exchange and removal by Anp32e reveals the importance of the nucleosome surface and nucleosome dynamics in processing the damaged chromatin template during DSB repair.The repair of DNA double-strand breaks (DSBs), which cleave the DNA backbone, requires remodeling of the local chromatin architecture. This reorganization of the chromatin is important for promoting access to the site of damage, for creating a template for the repair machinery, and for repackaging the chromatin and resetting the epigenetic landscape following repair. Chromatin remodeling at DSBs is linked to changes in posttranslational modification of histones. DSBs activate the ataxia-telangiectasia mutated (ATM) and DNA–PKcs kinases, which phosphorylate multiple DNA repair proteins, including histone H2AX. Phosphorylated H2AX (γH2AX) provides a binding site for mdc1, which promotes spreading of γH2AX for hundreds of kilobases either side of the break (1–3). DSBs also promote complex patterns of chromatin ubiquitination, including ubiquitination of H2A/H2AX by the RNF8/RNF168 ubiquitin ligases, which, in turn, creates binding sites for repair proteins such as 53BP1 and brca1 (4–7). DSBs also lead to increased methylation of histone H3 on lysine 36, which can regulate DNA repair pathway choice (8, 9) and methylation of H3 on lysine 9 (10), which drives activation of the Tip60 acetyltransferase and the ATM kinase (11, 12). Further, the NuA4–Tip60 complex (5, 13–15) promotes acetylation of histone H4 at DSBs and drives the formation of open, flexible chromatin domains at DSBs (5, 13, 14). The repair of DSBs is therefore fundamentally a chromatin-driven process requiring dynamic changes in histone modification and chromatin reorganization, which directly promote recruitment of DSB repair proteins (16).The NuA4–Tip60 remodeling complex plays a central role in nucleosome reorganization at DSBs (16). NuA4–Tip60 is a 16 subunit complex containing 2 key subunits—the p400 SWI/SNF ATPase and the Tip60 acetyltransferase. The p400 ATPase promotes exchange of H2A for the histone variant H2A.Z (13, 17). This increase in H2A.Z at DSBs then promotes acetylation of histone H4 by the Tip60, creating open, flexible chromatin at sites of DNA damage (5, 11, 14). Inactivation of NuA4–Tip60 blocks both H2A.Z exchange and H4 acetylation, leading to a reduction in chromatin mobility at DSBs. Consequently, loss of H2A.Z exchange leads to defective DSB repair, increased sensitivity to DNA damage, and genomic instability (13, 18, 19).Here, we demonstrate that H2A.Z exchange at DSBs is dynamic, with H2A.Z accumulating at DSBs within minutes of damage, followed by rapid H2A.Z eviction. Further, we show that Anp32e, an H2A.Z-specific histone chaperone, binds specifically to the docking domain of H2A.Z and is required to remove H2A.Z from the damaged chromatin template. Failure to remove H2A.Z leads to defects in DSB repair, including a loss of H4 acetylation, defects in nonhomologous end joining (NHEJ), and increased end resection of DSBs.     

N terminus of ASPP2 binds to Ras and enhances Ras/Raf/MEK/ERK activation to promote oncogene-induced senescence

The ASPP2 (also known as 53BP2L) tumor suppressor is a proapoptotic member of a family of p53 binding proteins that functions in part by enhancing p53-dependent apoptosis via its C-terminal p53-binding domain. Mounting evidence also suggests that ASPP2 harbors important nonapoptotic p53-independent functions. Structural studies identify a small G protein Ras-association domain in the ASPP2 N terminus. Because Ras-induced senescence is a barrier to tumor formation in normal cells, we investigated whether ASPP2 could bind Ras and stimulate the protein kinase Raf/MEK/ERK signaling cascade. We now show that ASPP2 binds to Ras–GTP at the plasma membrane and stimulates Ras-induced signaling and pERK1/2 levels via promoting Ras–GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation. These functions require the ASPP2 N terminus because BBP (also known as 53BP2S), an alternatively spliced ASPP2 isoform lacking the N terminus, was defective in binding Ras–GTP and stimulating Raf/MEK/ERK signaling. Decreased ASPP2 levels attenuated H-RasV12–induced senescence in normal human fibroblasts and neonatal human epidermal keratinocytes. Together, our results reveal a mechanism for ASPP2 tumor suppressor function via direct interaction with Ras–GTP to stimulate Ras-induced senescence in nontransformed human cells.ASPP2, also known as 53BP2L, is a tumor suppressor whose expression is altered in human cancers (1). Importantly, targeting of the ASPP2 allele in two different mouse models reveals that ASPP2 heterozygous mice are prone to spontaneous and γ-irradiation–induced tumors, which rigorously demonstrates the role of ASPP2 as a tumor suppressor (2, 3). ASPP2 binds p53 via the C-terminal ankyrin-repeat and SH3 domain (4–6), is damage-inducible, and can enhance damage-induced apoptosis in part through a p53-mediated pathway (1, 2, 7–10). However, it remains unclear what biologic pathways and mechanisms mediate ASPP2 tumor suppressor function (1). Indeed, accumulating evidence demonstrates that ASPP2 also mediates nonapoptotic p53-independent pathways (1, 3, 11–15).The induction of cellular senescence forms an important barrier to tumorigenesis in vivo (16–21). It is well known that oncogenic Ras signaling induces senescence in normal nontransformed cells to prevent tumor initiation and maintain complex growth arrest pathways (16, 18, 21–24). The level of oncogenic Ras activation influences its capacity to activate senescence; high levels of oncogenic H-RasV12 signaling leads to low grade tumors with senescence markers, which progress to invasive cancers upon senescence inactivation (25). Thus, tight control of Ras signaling is critical to ensure the proper biologic outcome in the correct cellular context (26–28).The ASPP2 C terminus is important for promoting p53-dependent apoptosis (7). The ASPP2 N terminus may also suppress cell growth (1, 7, 29–33). Alternative splicing can generate the ASPP2 N-terminal truncated protein BBP (also known as 53BP2S) that is less potent in suppressing cell growth (7, 34, 35). Although the ASPP2 C terminus mediates nuclear localization, full-length ASPP2 also localizes to the cytoplasm and plasma membrane to mediate extranuclear functions (7, 11, 12, 36). Structural studies of the ASPP2 N terminus reveal a β–Grasp ubiquitin-like fold as well as a potential Ras-binding (RB)/Ras-association (RA) domain (32). Moreover, ASPP2 can promote H-RasV12–induced senescence (13, 15). However, the molecular mechanism(s) of how ASPP2 directly promotes Ras signaling are complex and remain to be completely elucidated.Here, we explore the molecular mechanisms of how Ras-signaling is enhanced by ASPP2. We demonstrate that ASPP2: (i) binds Ras-GTP and stimulates Ras-induced ERK signaling via its N-terminal domain at the plasma membrane; (ii) enhances Ras-GTP loading and B-Raf/C-Raf dimerization and forms a ASPP2/Raf complex; (iii) stimulates Ras-induced C-Raf phosphorylation and activation; and (iv) potentiates H-RasV12–induced senescence in both primary human fibroblasts and neonatal human epidermal keratinocytes. These data provide mechanistic insight into ASPP2 function(s) and opens important avenues for investigation into its role as a tumor suppressor in human cancer.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

Mitigation of acute kidney injury by cell-cycle inhibitors that suppress both CDK4/6 and OCT2 functions

Acute kidney injury (AKI) is a potentially fatal syndrome characterized by a rapid decline in kidney function caused by ischemic or toxic injury to renal tubular cells. The widely used chemotherapy drug cisplatin accumulates preferentially in the renal tubular cells and is a frequent cause of drug-induced AKI. During the development of AKI the quiescent tubular cells reenter the cell cycle. Strategies that block cell-cycle progression ameliorate kidney injury, possibly by averting cell division in the presence of extensive DNA damage. However, the early signaling events that lead to cell-cycle activation during AKI are not known. In the current study, using mouse models of cisplatin nephrotoxicity, we show that the G1/S-regulating cyclin-dependent kinase 4/6 (CDK4/6) pathway is activated in parallel with renal cell-cycle entry but before the development of AKI. Targeted inhibition of CDK4/6 pathway by small-molecule inhibitors palbociclib (PD-0332991) and ribociclib (LEE011) resulted in inhibition of cell-cycle progression, amelioration of kidney injury, and improved overall survival. Of additional significance, these compounds were found to be potent inhibitors of organic cation transporter 2 (OCT2), which contributes to the cellular accumulation of cisplatin and subsequent kidney injury. The unique cell-cycle and OCT2-targeting activities of palbociclib and LEE011, combined with their potential for clinical translation, support their further exploration as therapeutic candidates for prevention of AKI.Cell division is a fundamental biological process that is tightly regulated by evolutionarily conserved signaling pathways (1, 2). The initial decision to start cell division, the fidelity of subsequent DNA replication, and the final formation of daughter cells is monitored and regulated by these essential pathways (2–6). The cyclin-dependent kinases (CDKs) are the central players that orchestrate this orderly progression through the cell cycle (1, 2, 6, 7). The enzymatic activity of CDKs is regulated by complex mechanisms that include posttranslational modifications and expression of activating and inhibitory proteins (1, 2, 6, 7). The spatial and temporal changes in the activity of these CDK complexes are thought to generate the distinct substrate specificities that lead to sequential and unidirectional progression of the cell cycle (1, 8, 9).Cell-cycle deregulation is a universal feature of human cancer and a long-sought-after target for anticancer therapy (1, 10–13). Frequent genetic or epigenetic changes in mitogenic pathways, CDKs, cyclins, or CDK inhibitors are observed in various human cancers (1, 4, 11). In particular, the G1/S-regulating CDK4/6–cyclin D–inhibitors of CDK4 (INK4)–retinoblastoma (Rb) protein pathway frequently is disrupted in cancer cells (11, 14). These observations provided an impetus to develop CDK inhibitors as anticancer drugs. However, the earlier class of CDK inhibitors had limited specificity, inadequate clinical activity, poor pharmacokinetic properties, and unacceptable toxicity profiles (10, 11, 14, 15). These disappointing initial efforts now have been followed by the development of the specific CDK4/6 inhibitors palbociclib (PD0332991), ribociclib (LEE011), and abemaciclib (LY2835219), which have demonstrated manageable toxicities, improved pharmacokinetic properties, and impressive antitumor activity, especially in certain forms of breast cancer (14, 16). Successful early clinical trials with these three CDK4/6 inhibitors have generated cautious enthusiasm that these drugs may emerge as a new class of anticancer agents (14, 17). Palbociclib recently was approved by Food and Drug Administration for the treatment of metastatic breast cancer and became the first CDK4/6 inhibitor approved for anticancer therapy (18).In addition to its potential as an anticancer strategy, CDK4/6 inhibition in normal tissues could be exploited therapeutically for wide-ranging clinical conditions. For example, radiation-induced myelosuppression, caused by cell death of proliferating hematopoietic stem/progenitor cells, can be rescued by palbociclib (19, 20). Furthermore, cytotoxic anticancer agents cause significant toxicities to normal proliferating cells, which possibly could be mitigated by the concomitant use of CDK4/6 inhibitors (20, 21). More broadly, cell-cycle inhibition could have beneficial effects in disorders in which maladaptive proliferation of normal cells contributes to the disease pathology, as observed in vascular proliferative diseases, hyperproliferative skin diseases, and autoimmune disorders (22, 23). In support of this possibility, palbociclib treatment recently was reported to ameliorate disease progression in animal models of rheumatoid arthritis through cell-cycle inhibition of synovial fibroblasts (24).Abnormal cellular proliferation also is a hallmark of various kidney diseases (25), and cell-cycle inhibition has been shown to ameliorate significantly the pathogenesis of polycystic kidney disease (26), nephritis (27), and acute kidney injury (AKI) (28). Remarkably, during AKI, the normally quiescent renal tubular cells reenter the cell cycle (29–34), and blocking cell-cycle progression can reduce renal injury (28). Here, we provide evidence that the CDK4/6 pathway is activated early during AKI and demonstrate significant protective effects of CDK4/6 inhibitors in animal models of cisplatin-induced AKI. In addition, we found that the CDK4/6 inhibitors palbociclib and LEE011 are potent inhibitors of organic cation transporter 2 (OCT2), a cisplatin uptake transporter highly expressed in renal tubular cells (35–37). Our findings provide a rationale for the clinical development of palbociclib and LEE011 for the prevention and treatment of AKI.     

Hierarchical looping of zigzag nucleosome chains in metaphase chromosomes

The architecture of higher-order chromatin in eukaryotic cell nuclei is largely unknown. Here, we use electron microscopy-assisted nucleosome interaction capture (EMANIC) cross-linking experiments in combination with mesoscale chromatin modeling of 96-nucleosome arrays to investigate the internal organization of condensed chromatin in interphase cell nuclei and metaphase chromosomes at nucleosomal resolution. The combined data suggest a novel hierarchical looping model for chromatin higher-order folding, similar to rope flaking used in mountain climbing and rappelling. Not only does such packing help to avoid tangling and self-crossing, it also facilitates rope unraveling. Hierarchical looping is characterized by an increased frequency of higher-order internucleosome contacts for metaphase chromosomes compared with chromatin fibers in vitro and interphase chromatin, with preservation of a dominant two-start zigzag organization associated with the 30-nm fiber. Moreover, the strong dependence of looping on linker histone concentration suggests a hierarchical self-association mechanism of relaxed nucleosome zigzag chains rather than longitudinal compaction as seen in 30-nm fibers. Specifically, concentrations lower than one linker histone per nucleosome promote self-associations and formation of these looped networks of zigzag fibers. The combined experimental and modeling evidence for condensed metaphase chromatin as hierarchical loops and bundles of relaxed zigzag nucleosomal chains rather than randomly coiled threads or straight and stiff helical fibers reconciles aspects of other models for higher-order chromatin structure; it constitutes not only an efficient storage form for the genomic material, consistent with other genome-wide chromosome conformation studies that emphasize looping, but also a convenient organization for local DNA unraveling and genome access.The physical packaging of megabase pairs of genomic DNA stored as the chromatin fiber in eukaryotic cell nuclei has been one of the great challenges in biology (1). The limited resolution and disparate levels that can be studied by both experimental and modeling studies of chromatin, which exhibits multiple spatial and temporal scales par excellence, make it challenging to present an integrated structural view, from nucleosomes to chromosomes (2). Because all fundamental template-directed processes of DNA depend on chromatin architecture, advances in our understanding of chromatin higher-order organization are needed to help interpret numerous regulatory events from DNA damage repair to epigenetic control.At the primary structural level, the DNA makes ∼1.7 left-superhelical turns around eight core histones to form a nucleosome core. The nucleosome cores are connected by linker DNA to form nucleosome arrays. An X-ray crystal structure of the nucleosome core has been solved at atomic resolution (3), and a short, four-nucleosome array has also been solved (4). Next, at the secondary structural level, the nucleosome arrays, aided by linker histones (H1 or H5), form a compact chromatin fiber with a diameter of ∼30 nm and longitudinal compaction of 5–7 nucleosomes per 11 nm (5–8). However, evidence for 30-nm fibers in interphase nuclei of living cells has been controversial (reviewed in refs. 9 and 10). For example, whereas a distinct 30-nm fiber architecture is observed in terminally differentiated cells (11, 12), neither continuous nor periodic 30-nm fibers are observed in the nuclei of proliferating cells (13–15). However, zigzag features of the chromatin fibers are well supported by nucleosome interaction mapping in vitro (16) and in vivo (15).For chromatin architecture within metaphase chromosomes, fluorescence studies of mitotic chromosome condensation in vivo (17), cryo-EM observations of unfixed and unstained chromosomes in situ (18), and small-angle X-ray scattering (19) show no structures resembling folded 30-nm fibers and instead suggest random folding of soft polymers. Evidence is also accumulating that during chromosome condensation in mitosis, chromatin higher-order structure is dramatically altered at the global level (20) by significant increase in looping (21). A random type of looping, however, cannot explain sharp chromosomal boundaries separating the translocated genomic regions in metaphase chromosomes (22) as well as formation of highly localized fibers of transgenic DNA, up to 250 nm in diameter, detected by fluorescence imaging in vivo (17). In contrast, a hierarchical or layered looping could explain the above aspects of chromosome organization; in addition, it could help reconcile the experiments in living cells with in vitro data and determine which aspects of the secondary structure are retained in the metaphase chromosome and how these features correlate with the polymer melt model (18, 23).Here we apply the EM-assisted nucleosome interaction capture (EMANIC) technique, which captures nearest-neighbor interactions in combination with mesoscale modeling of chromatin fibers (16) to deduce chromatin architecture in interphase nuclei and metaphase chromosomes. Our results reveal persistence of the zigzag geometry as a dominant architectural motif in these types of chromatin. For metaphase chromosomes, we report a dramatic increase in longer-range interactions, consistent with intrafiber looping, quite different from that seen in compact chromatin fibers in vitro and interphase chromatin in vivo. Modeling also shows hierarchical looping for long fibers, with the loop occurrence strongly modulated by the density of linker histones. Such looping of loosely folded zigzag arrays appears to be an efficient mechanism for both condensing and unraveling the genomic material. Our hierarchical looping mechanism can also explain how distant regulatory DNA sites can be brought together naturally for genic interactions and how linker histone levels and epigenetic histone modifications can further modulate global and local chromatin architecture.