Modeling phenotypic heterogeneity towards evolutionarily inspired osteosarcoma therapy.

Osteosarcoma is the most common bone sarcoma in children and young adults. While universally delivered, chemotherapy only benefits roughly half of patients with localized disease. Increasingly, intratumoral heterogeneity is recognized as a source of therapeutic resistance. In this study, we develop and evaluate an in vitro model of osteosarcoma heterogeneity based on phenotype and genotype. Cancer cell populations vary in their environment-specific growth rates and in their sensitivity to chemotherapy. We present the genotypic and phenotypic characterization of an osteosarcoma cell line panel with a focus on co-cultures of the most phenotypically divergent cell lines, 143B and SAOS2. Modest environmental (pH, glutamine) or chemical perturbations dramatically shift the success and composition of cell lines. We demonstrate that in nutrient rich culture conditions 143B outcompetes SAOS2. But, under nutrient deprivation or conventional chemotherapy, SAOS2 growth can be favored in spheroids. Importantly, when the simplest heterogeneity state is evaluated, a two-cell line coculture, perturbations that affect the faster growing cell line have only a modest effect on final spheroid size. Thus the only evaluated therapies to eliminate the spheroids were by switching therapies from a first strike to a second strike. This extensively characterized, widely available system, can be modeled and scaled to allow for improved strategies to anticipate resistance in osteosarcoma due to heterogeneity.

Identification of Selective ATP-Competitive CMG Helicase Inhibitors for Cancer Intervention that Disrupt CMG-Replisome Function.

The human CMG helicase (Cdc45-MCM-GINS) is a novel target for anti-cancer therapy due to tumor-specific weaknesses in CMG function induced by oncogenic changes and the need for CMG function during recovery from replicative stresses such as chemotherapy. Here, we developed an orthogonal biochemical screening approach and identified selective CMG inhibitors (CMGi) that inhibit ATPase and helicase activities in an ATP-competitive manner at low micromolar concentrations. Structure-activity information and in silico docking indicate that CMGi occupy ATP binding sites and channels within MCM subunits leading to the ATP clefts, which are likely used for ATP/ADP ingress or egress. CMGi inhibit cell growth and DNA replication using multiple molecular mechanisms. CMGi block helicase assembly steps that require ATP binding/hydrolysis by the MCM complex, specifically MCM ring assembly on DNA and GINS recruitment to DNA-loaded MCM hexamers. During S-phase, inhibition of MCM ATP binding/hydrolysis by CMGi causes a 'reverse allosteric' dissociation of Cdc45/GINS from the CMG that destabilizes the replisome and disrupts interactions with Ctf4, Mcm10, and DNA polymerase-α, -δ, -ε, resulting in DNA damage. These novel CMGi are selectively toxic toward tumor cells and define a new class of CMG helicase-targeted anti-cancer compounds with distinct mechanisms of action.

The CMG helicase and cancer: a tumor "engine" and weakness with missing mutations.

The replicative Cdc45-MCM-GINS (CMG) helicase is a large protein complex that functions in the DNA melting and unwinding steps as a component of replisomes during DNA replication in mammalian cells. Although the CMG performs this important role in cell growth, the CMG is not a simple bystander in cell cycle events. Components of the CMG, specifically the MCM precursors, are also involved in maintaining genomic stability by regulating DNA replication fork speeds, facilitating recovery from replicative stresses, and preventing consequential DNA damage. Given these important functions, MCM/CMG complexes are highly regulated by growth factors such as TGF-ß1 and by signaling factors such as Myc, Cyclin E, and the retinoblastoma protein. Mismanagement of MCM/CMG complexes when these signaling mediators are deregulated, and in the absence of the tumor suppressor protein p53, leads to increased genomic instability and is a contributor to tumorigenic transformation and tumor heterogeneity. The goal of this review is to provide insight into the mechanisms and dynamics by which the CMG is regulated during its assembly and activation in mammalian genomes, and how errors in CMG regulation due to oncogenic changes promote tumorigenesis. Finally, and most importantly, we highlight the emerging understanding of the CMG helicase as an exploitable vulnerability and novel target for therapeutic intervention in cancer.

Myc and the Replicative CMG Helicase: The Creation and Destruction of Cancer: Myc Over-Activation of CMG Helicases Drives Tumorigenesis and Creates a Vulnerability in CMGs for Therapeutic Intervention.

Myc-driven tumorigenesis involves a non-transcriptional role for Myc in over-activating replicative Cdc45-MCM-GINS (CMG) helicases. Excessive stimulation of CMG helicases by Myc mismanages CMG function by diminishing the number of reserve CMGs necessary for fidelity of DNA replication and recovery from replicative stresses. One potential outcome of these events is the creation of DNA damage that alters genomic structure/function, thereby acting as a driver for tumorigenesis and tumor heterogeneity. Intriguingly, another potential outcome of this Myc-induced CMG helicase over-activation is the creation of a vulnerability in cancer whereby tumor cells specifically lack enough unused reserve CMG helicases to recover from fork-stalling drugs commonly used in chemotherapy. This review provides molecular and clinical support for this provocative hypothesis that excessive activation of CMG helicases by Myc may not only drive tumorigenesis, but also confer an exploitable "reserve CMG helicase vulnerability" that supports developing innovative CMG-focused therapeutic approaches for cancer management.

Myc-driven chromatin accessibility regulates Cdc45 assembly into CMG helicases.

Myc-driven tumorigenesis involves a non-transcriptional role for Myc in over-activating replication origins. We show here that the mechanism underlying this process involves a direct role for Myc in activation of Cdc45-MCM-GINS (CMG) helicases at Myc-targeted sites. Myc induces decondensation of higher-order chromatin at targeted sites and is required for chromatin access at a chromosomal origin. Myc-driven chromatin accessibility promotes Cdc45/GINS recruitment to resident MCMs, and activation of CMGs. Myc-Box II, which is necessary for Myc-driven transformation, is required for Myc-induced chromatin accessibility, Cdc45/GINS recruitment, and replication stimulation. Myc interactors GCN5, Tip60, and TRRAP are essential for chromatin unfolding and recruitment of Cdc45, and co-expression of GCN5 or Tip60 with MBII-deficient Myc rescues these events and promotes CMG activation. Finally, Myc and Cdc45 interact and physiologic conditions for CMG assembly require the functions of Myc, MBII, and GCN5 for Cdc45 recruitment and initiation of DNA replication.

TGFß1 Cell Cycle Arrest Is Mediated by Inhibition of MCM Assembly in Rb-Deficient Conditions.

Transforming growth factor ß1 (TGFß1) is a potent inhibitor of cell growth that targets gene-regulatory events, but also inhibits the function of CDC45-MCM-GINS helicases (CMG; MCM, Mini-Chromosome Maintenance; GINS, Go-Ichi-Ni-San) through multiple mechanisms to achieve cell-cycle arrest. Early in G1, TGFß1 blocks MCM subunit expression and suppresses Myc and Cyclin E/Cdk2 activity required for CMG assembly, should MCMs be expressed. Once CMGs are assembled in late-G1, TGFß1 blocks CMG activation using a direct mechanism involving the retinoblastoma (Rb) tumor suppressor. Here, in cells lacking Rb, TGFß1 does not suppress Myc, Cyclin E/Cdk2 activity, or MCM expression, yet growth arrest remains intact and Smad2/3/4-dependent. Such arrest occurs due to inhibition of MCM hexamer assembly by TGFß1, which is not seen when Rb is present and MCM subunit expression is normally blocked by TGFß1. Loss of Smad expression prevents TGFß1 suppression of MCM assembly. Mechanistically, TGFß1 blocks a Cyclin E-Mcm7 molecular interaction required for MCM hexamer assembly upstream of CDC10-dependent transcript-1 (CDT1) function. Accordingly, overexpression of CDT1 with an intact MCM-binding domain abrogates TGFß1 arrest and rescues MCM assembly. The ability of CDT1 to restore MCM assembly and allow S-phase entry indicates that, in the absence of Rb and other canonical mediators, TGFß1 relies on inhibition of Cyclin E-MCM7 and MCM assembly to achieve cell cycle arrest. IMPLICATIONS: These results demonstrate that the MCM assembly process is a pivotal target of TGFß1 in eliciting cell cycle arrest, and provide evidence for a novel oncogenic role for CDT1 in abrogating TGFß1 inhibition of MCM assembly.

DNase I Chromatin Accessibility Analysis.

Chromatin consists of compacted DNA in complex with proteins and contributes to the organization of DNA and its stability. Furthermore, chromatin plays key roles in regulating cellular processes such as DNA replication, transcription, DNA repair, and mitosis. Chromatin assumes more compact (inaccessible) or decondensed (accessible) conformations depending on the function that is being supported in the genome, either locally or globally. The activity of nucleases has been used previously to assess the accessibility of specific genomic regions in vitro, such as origins of replication at varying points in the cell cycle. Here, we provide an assay to determine the accessibility of specific human genomic regions (example used herein: Lamin B2 origin of DNA replication) by measuring the effect of DNase I nuclease on qPCR signal from the studied site. This assay provides a powerful method to interrogate the molecular mechanisms that regulate chromatin accessibility, and how these processes affect various cellular functions involving the human genome that require manipulation of chromatin conformation.

Data supporting the functional role of Eleven-nineteen Lysine-rich Leukemia 3 (ELL3) in B cell lymphoma cell line cells.

The data presented here are related to the research article entitled "Selective expression of the transcription elongation factor ELL3 in B cells prior to ELL2 drives proliferation and survival" (Alexander et al., 2017) [1]. The cited research article characterizes Eleven-nineteen Lysine-rich Leukemia 3 (ELL3) expression in the B cell compartment and functional dependence in B lymphoma cell lines. This data report describes the mRNA expression pattern in a panel of cell lines representing the B cell compartment, supplementing the protein expression data presented in the associated research report. In addition, a reanalysis is presented of publicly available mRNA expression data from primary murine B cells to reveal dynamic regulation of the ELL family members post LPS stimulation (Barwick et al., 2016) [2]. The effect of ELL3 depletion on cell morphology, latent Epstein Barr Virus (EBV) lytic replication and differentiation markers in a Burkitt's lymphoma (BL) cell line cells are presented.

Selective expression of the transcription elongation factor ELL3 in B cells prior to ELL2 drives proliferation and survival.

B cell activation is dependent on a large increase in transcriptional output followed by focused expression on secreted immunoglobulin as the cell transitions to an antibody producing plasma cell. The rapid transcriptional induction is facilitated by the release of poised RNA pol II into productive elongation through assembly of the super elongation complex (SEC). We report that a SEC component, the Eleven -nineteen Lysine-rich leukemia (ELL) family member 3 (ELL3) is dynamically up-regulated in mature and activated human B cells followed by suppression as B cells transition to plasma cells in part mediated by the transcription repressor PRDM1. Burkitt's lymphoma and a sub-set of Diffuse Large B cell lymphoma cell lines abundantly express ELL3. Depletion of ELL3 in the germinal center derived lymphomas results in severe disruption of DNA replication and cell division along with increased DNA damage and cell death. This restricted utilization and survival dependence reveal a key step in B cell activation and indicate a potential therapeutic target against B cell lymphoma's with a germinal center origin.

The N Terminus of the Retinoblastoma Protein Inhibits DNA Replication via a Bipartite Mechanism Disrupted in Partially Penetrant Retinoblastomas.

The N-terminal domain of the retinoblastoma (Rb) tumor suppressor protein (RbN) harbors in-frame exon deletions in partially penetrant hereditary retinoblastomas and is known to impair cell growth and tumorigenesis. However, how such RbN deletions contribute to Rb tumor- and growth-suppressive functions is unknown. Here we establish that RbN directly inhibits DNA replication initiation and elongation using a bipartite mechanism involving N-terminal exons lost in cancer. Specifically, Rb exon 7 is necessary and sufficient to target and inhibit the replicative CMG helicase, resulting in the accumulation of inactive CMGs on chromatin. An independent N-terminal loop domain, which forms a projection, specifically blocks DNA polymerase α (Pol-α) and Ctf4 recruitment without affecting DNA polymerases ε and δ or the CMG helicase. Individual disruption of exon 7 or the projection in RbN or Rb, as occurs in inherited cancers, partially impairs the ability of Rb/RbN to inhibit DNA replication and block G1-to-S cell cycle transit. However, their combined loss abolishes these functions of Rb. Thus, Rb growth-suppressive functions include its ability to block replicative complexes via bipartite, independent, and additive N-terminal domains. The partial loss of replication, CMG, or Pol-α control provides a potential molecular explanation for how N-terminal Rb loss-of-function deletions contribute to the etiology of partially penetrant retinoblastomas.

Suppression of Reserve MCM Complexes Chemosensitizes to Gemcitabine and 5-Fluorouracil.

UNLABELLED: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of cancer and is very difficult to treat with conventional chemotherapeutic regimens. Gemcitabine and 5-fluorouracil are used in the management of PDAC and act by indirectly blocking replicative forks. However, these drugs are not highly effective at suppressing disease progression, indicating a need for the development of innovative therapeutic approaches. Recent studies indicate that suppression of the MCM helicase may provide a novel means to sensitize cancer cells to chemotherapeutic agents that inhibit replicative fork progression. Mammalian cells assemble more MCM complexes on DNA than are required to start S-phase. The excess MCM complexes function as backup initiation sites under conditions of replicative stress. The current study provides definitive evidence that cosuppression of the excess/backup MCM complexes sensitizes PDAC tumor lines to both gemcitabine and 5-FU, leading to increased loss of proliferative capacity compared with drugs alone. This occurs because reduced MCM levels prevent efficient recovery of DNA replication in tumor cells exposed to drug. PDAC tumor cells are more sensitive to MCM loss in the presence of gemcitabine than are nontumor, immortalized epithelial cells. Similarly, colon tumor cells are rendered less viable when cosuppression of MCM complexes occurs during exposure to the crosslinking agent oxaliplatin or topoisomerase inhibitor etoposide. IMPLICATIONS: These studies demonstrate that suppressing the backup complement of MCM complexes provides an effective sensitizing approach with the potential to increase the therapeutic index of drugs used in the clinical management of PDAC and other cancers.

Analysis of DNA replication associated chromatin decondensation: in vivo assay for understanding chromatin remodeling mechanisms of selected proteins.

Of critical importance to many of the events underlying transcriptional control of gene expression are modifications to core and linker histones that regulate the accessibility of trans-acting factors to the DNA substrate within the context of chromatin. Likewise, control over the initiation of DNA replication, as well as the ability of the replication machinery to proceed during elongation through the multiple levels of chromatin condensation that are likely to be encountered, is known to involve the creation of chromatin accessibility. In the latter case, chromatin access will likely need to be a transient event so as to prevent total genomic unraveling of the chromatin that would be deleterious to cells. While there are many molecular and biochemical approaches in use to study histone changes and their relationship to transcription and chromatin accessibility, few techniques exist that allow a molecular dissection of the events underlying DNA replication control as it pertains to chromatin changes and accessibility. Here, we outline a novel experimental strategy for addressing the ability of specific proteins to induce large-scale chromatin unfolding (decondensation) in vivo upon site-specific targeting to an engineered locus. Our laboratory has used this powerful system in novel ways to directly address the ability of DNA replication proteins to create chromatin accessibility, and have incorporated modifications to the basic approach that allow for a molecular genetic analysis of the mechanisms and associated factors involved in causing chromatin decondensation by a protein of interest. Alternative approaches involving co-expression of other proteins (competitors or stimulators), concurrent drug treatments, and analysis of co-localizing histone modifications are also addressed, all of which are illustrative of the utility of this experimental system for extending basic findings to physiologically relevant mechanisms. Although used by our group to analyze mechanisms underlying DNA replication associated chromatin accessibility, this unique and powerful experimental system has the propensity to be a valuable tool for understanding chromatin remodeling mechanisms orchestrated by other cellular processes such as DNA repair, recombination, mitotic chromosome condensation, or other chromosome dynamics involving chromatin alterations and accessibility.

Chemoprevention Trial Feasibility Using Botanicals in Exceptionally High Risk Populations for Lung Cancer.

While chemoprevention with botanicals shows promise in reducing cancer risk, recruitment and retention of participants for trials continues to be costly and presents unique challenges. Knowledge of interest, willingness of target populations and evaluation of design challenges are critical to improve accrual in these chemoprevention trials. OBJECTIVE: The study assessed interest and willingness of former smokers to participate in a chemoprevention trial using a botanical agent. METHODS: An introductory letter and survey instrument were mailed to 609 consecutive, former heavy smokers, with no cancer, from a database of 826 subjects at the Moffitt Cancer Center. RESULTS: 202 (40.4%) subjects returned completed surveys. 92-96% reported interest in receiving free lung exams and knowing their lung cancer risk. 88% were interested in participating in a trial evaluating a botanical agent for lung cancer prevention. Over 92% of subjects reported willingness to comply with study requirements; multiple blood draws and trips to the Center, spiral CTs and chest x-rays. Subjects were relatively less enthusiastic (73-79%) about bronchoscopy, taking multiple study agents and assignment to placebo arm. CONCLUSIONS: Our study strongly suggests feasibility, highlights potential challenges and the significant interest and willingness of this exceptionally high risk population to participate in chemoprevention trials.

Phase 2 randomized study of enzastaurin (LY317615) for lung cancer prevention in former smokers.

BACKGROUND: Chemoprevention for lung cancer with nutraceutical or anti-inflammatory agents has had mixed clinical benefit. Novel targeted agents hold the promise of greater efficacy and selectivity. The authors of this report evaluated enzastaurin, a selective protein kinase C-ß (PKC-ß) inhibitor with antiproliferative and proapoptotic properties, in former smokers. METHODS: The primary objective of this study was to compare the average fraction of Ki-67-stained cells (the Ki-67 labeling index [LI]) in bronchial biopsy specimens that were collected before and after treatment. Participants were randomized (2:1) to receive either 6 months of daily oral enzastaurin (500 mg) or placebo. Stratification was based on morphology, history of lung cancer, and airway obstruction. RESULTS: In pretrial investigations, the rationale for PKC-ß inhibition and pathway interrogation was established in premalignant lesions and early stage lung cancer. In an intent-to-treat analysis, of 40 randomized participants, there was no significant difference in the pretreatment/post-treatment change in the Ki-67 LI between the enzastaurin group and the placebo group (P = .53). Six participants discontinued enzastaurin, including 4 participants who had adverse events, including abdominal distension, deep vein thrombosis, hyponatremia, and rash, and 2 participants who decided to discontinue. One participant in the placebo group was discontinued on the study because of noncompliance. Two participants had ≥1 serious adverse event (bradycardia, deep vein thrombosis, and hypotension). CONCLUSIONS: To the authors' knowledge, this represents the first chemoprevention trial with a non-US Food and Drug Administration-approved, oral, small-molecule-targeted agent. Although the primary endpoint was not met, enzastaurin was tolerable for 6 months by 75% of participants, and there was a suggestion of response in a subset analysis that was restricted to those who had metaplastic or dysplastic lesions.

Curcumin: a novel Stat3 pathway inhibitor for chemoprevention of lung cancer.

Multiple studies from independent groups find evidence for signal transducer and activator of transcription 3 (Stat3) activation in nearly 50% of lung cancers, suggesting a functional role for this target in subsets of lung cancer. On the basis of the existing evidence, we hypothesized that bioavailable curcuminoid complex may modulate lung carcinogenesis, primarily by inhibiting Stat3 activation. With the safety of this being botanically well established, the objective of these studies was to test our hypothesis in vitro and in vivo in an effort to inform the design of a phase II chemoprevention trial in former smokers. We treated non-tumor-derived, normal (but immortalized) human bronchial epithelial cells (AALE) (Lundberg et al., 2002; Pillai et al., 2011) and lung adenocarcinoma-derived cells (H441) with bioactive curcumin C3 complex. Asynchronous cells in each case were treated with curcumin for 24 h, followed by immunoblotting for Stat3 and activated Stat3-P, prior signal of which was used for normalization. We also completed a preclinical trial in which 12 mice were randomly divided into three groups and subjected to 3 days or 9 days of curcumin intraperitoneal injections, followed by analysis of lung tissues for Stat3-P changes and growth suppressive effects of the curcumin. The growth suppressive effects were measured using Cyclin D1 and the replicative helicase subunit, Mcm2, as surrogates for the proliferative capacity of the tissues. In-vitro studies with curcuminoid complex demonstrated that the activity of Stat3 in both normal bronchoepithelial cells and lung cancer-derived cells is sensitive to curcumin exposure. In a dose-dependent manner, curcumin treatment resulted in significant suppression of Stat3 phosphorylation and reduction in the proliferative capacity of both cell types. In the preclinical trial with rodent models, curcumin reduced Stat3-P and the proliferative markers CycD1 and Mcm2 in mice lung tissues in vivo. These culture and preclinical studies indicate that the activity of the Stat3 pathway can be suppressed by curcumin treatment, concomitant with a reduction in cell proliferation, supporting our hypothesis that inhibition of the Stat3 pathway represents at least one important mechanism by which curcumin elicits its effects on the bronchoepithelium. These data provide a rationale for the use of curcumin as a promising chemopreventive agent in high-risk populations such as former smokers.

Cdc45 limits replicon usage from a low density of preRCs in mammalian cells.

Little is known about mammalian preRC stoichiometry, the number of preRCs on chromosomes, and how this relates to replicon size and usage. We show here that, on average, each 100-kb of the mammalian genome contains a preRC composed of approximately one ORC hexamer, 4-5 MCM hexamers, and 2 Cdc6. Relative to these subunits, ∼0.35 total molecules of the pre-Initiation Complex factor Cdc45 are present. Thus, based on ORC availability, somatic cells contain ∼70,000 preRCs of this average total stoichiometry, although subunits may not be juxtaposed with each other. Except for ORC, the chromatin-bound complement of preRC subunits is even lower. Cdc45 is present at very low levels relative to the preRC subunits, but is highly stable, and the same limited number of stable Cdc45 molecules are present from the beginning of S-phase to its completion. Efforts to artificially increase Cdc45 levels through ectopic expression block cell growth. However, microinjection of excess purified Cdc45 into S-phase nuclei activates additional replication foci by three-fold, indicating that Cdc45 functions to activate dormant preRCs and is rate-limiting for somatic replicon usage. Paradoxically, although Cdc45 colocalizes in vivo with some MCM sites and is rate-limiting for DNA replication to occur, neither Cdc45 nor MCMs colocalize with active replication sites. Embryonic metazoan chromatin consists of small replicons that are used efficiently via an excess of preRC subunits. In contrast, somatic mammalian cells contain a low density of preRCs, each containing only a few MCMs that compete for limiting amounts of Cdc45. This provides a molecular explanation why, relative to embryonic replicon dynamics, somatic replicons are, on average, larger and origin efficiency tends to be lower. The stable, continuous, and rate-limiting nature of Cdc45 suggests that Cdc45 contributes to the staggering of replicon usage throughout S-phase, and that replicon activation requires reutilization of existing Cdc45 during S-phase.

Cell cycle arrest by transforming growth factor beta1 near G1/S is mediated by acute abrogation of prereplication complex activation involving an Rb-MCM interaction.

Understanding inhibitory mechanisms of transforming growth factor beta1 (TGF-beta1) has provided insight into cell cycle regulation and how TGF-beta1 sensitivity is lost during tumorigenesis. We show here that TGF-beta1 utilizes a previously unknown mechanism targeting the function of prereplication complexes (pre-RCs) to acutely block S-phase entry when added to cells in late G(1), after most G(1) events have occurred. TGF-beta1 treatment in early G(1) suppresses Myc and CycE-Cdk2 and blocks pre-RC assembly. However, TGF-beta1 treatment in late G(1) acutely blocks S-phase entry by inhibiting activation of fully assembled pre-RCs, with arrest occurring prior to the helicase unwinding step at G(1)/S. This acute block by TGF-beta1 requires the function of Rb in late G(1) but does not involve Myc/CycE-Cdk2 suppression or transcriptional control. Instead, Rb mediates TGF-beta1 late-G(1) arrest by targeting the MCM helicase. Rb binds the MCM complex during late G(1) via a direct interaction with Mcm7, and TGF-beta1 blocks their dissociation at G(1)/S. Loss of Rb or overexpression of Mcm7 or its Rb-binding domain alone abrogates late-G(1) arrest by TGF-beta1. These results demonstrate that TGF-beta1 acutely blocks entry into S phase by inhibiting pre-RC activation and suggest a novel role for Rb in mediating this effect of TGF-beta1 through direct interaction with and control of the MCM helicase.

Mammalian MCM loading in late-G(1) coincides with Rb hyperphosphorylation and the transition to post-transcriptional control of progression into S-phase.

BACKGROUND: Control of the onset of DNA synthesis in mammalian cells requires the coordinated assembly and activation of the pre-Replication Complex. In order to understand the regulatory events controlling preRC dynamics, we have investigated how the timing of preRC assembly relates temporally to other biochemical events governing progress into S-phase. METHODOLOGY/PRINCIPAL FINDING: In murine and Chinese hamster (CHO) cells released from quiescence, the loading of the replicative MCM helicase onto chromatin occurs in the final 3-4 hrs of G(1). Cdc45 and PCNA, both of which are required for G(1)-S transit, bind to chromatin at the G(1)-S transition or even earlier in G(1), when MCMs load. An RNA polymerase II inhibitor (DRB) was added to synchronized murine keratinocytes to show that they are no longer dependent on new mRNA synthesis 3-4 hrs prior to S-phase entry, which is also true for CHO and human cells. Further, CHO cells can progress into S-phase on time, and complete S-phase, under conditions where new mRNA synthesis is significantly compromised, and such mRNA suppression causes no adverse effects on preRC dynamics prior to, or during, S-phase progression. Even more intriguing, hyperphosphorylation of Rb coincides with the start of MCM loading and, paradoxically, with the time in late-G(1) when de novo mRNA synthesis is no longer rate limiting for progression into S-phase. CONCLUSIONS/SIGNIFICANCE: MCM, Cdc45, and PCNA loading, and the subsequent transit through G(1)-S, do not depend on concurrent new mRNA synthesis. These results indicate that mammalian cells pass through a distinct transition in late-G(1) at which time Rb becomes hyperphosphorylated and MCM loading commences, but that after this transition the control of MCM, Cdc45, and PCNA loading and the onset of DNA replication are regulated at the post-transcriptional level.

In vivo chromatin decondensation assays: molecular genetic analysis of chromatin unfolding characteristics of selected proteins.

Of critical importance to many of the events underlying transcriptional control of gene expression are modifications to core and linker histones that regulate the accessibility of trans-acting factors to the DNA substrate within the context of chromatin. Likewise, control over the initiation of DNA replication, as well as the ability of the replication machinery to proceed during elongation through the multiple levels of chromatin condensation that are likely to be encountered, is almost certain to involve the creation of chromatin accessibility. In the latter case in particular, chromatin access will likely need to be a transient event so as to prevent total genomic unraveling of the chromatin that would be deleterious to cells. While there are many molecular and biochemical approaches in use to study histone changes and their relationship to transcription and chromatin accessibility, few techniques exist that allow a molecular dissection of the events underlying DNA replication control as it pertains to chromatin changes and accessibility. In this review, we outline a novel experimental strategy for addressing the ability of specific proteins to induce large-scale chromatin unfolding (decondensation) in vivo upon site-specific targeting to an engineered locus. We have used this system successfully to directly address the ability of DNA replication proteins to create chromatin accessibility and have incorporated modifications to the basic approach that allow for a molecular genetic analysis of the players involved in causing chromatin decondensation by a protein of interest. Here, we briefly describe the nature of the experimental system, its history, and a basic protocol for using the system. Alternative approaches involving co-transfections, concurrent drug treatments, and analysis of co-localizing histone modifications are also addressed, which are useful for extending basic findings to physiological mechanisms.